Inhibitors of prenyl-protein transferase

ABSTRACT

The present invention comprises piperazine/piperazinone-containing compounds having multicyclic ring system substituents on one of the piperazine/piperazinone nitrogens, which inhibit prenyl-protein transferases, including farnesyl-protein transferase and geranylgeranyl-protein transferase type I. Such therapeutic compounds are useful in the treatment of cancer.

RELATED APPLICATION

The present patent application claims the benefit of provisionalapplication Ser. No. 60/127,259, filed Mar. 31, 1999, and provisionalapplication Ser. No. 60/122,970, filed Mar. 3, 1999, both of which werepending on the date of the filing of the present invention.

BACKGROUND OF THE INVENTION

The present invention relates to certain compounds that are useful forthe inhibition of prenyl-protein transferases and the treatment ofcancer. In particular, the invention relates to prenyl-proteintransferase inhibitors which are efficacious in vivo as inhibitors ofgeranylgeranyl-protein transferase type I (GGTase-I) and that inhibitthe cellular processing of both the H-Ras protein and the K4B-Rasprotein.

Prenylation of proteins by prenyl-protein transferases represents aclass of post-translational modification (Glomset, J. A., Gelb, M. H.,and Farnsworth, C. C. (1990). Trends Biochem. Sci. 15, 139-142; Maltese,W. A. (1990). FASEB J. 4, 3319-3328). This modification typically isrequired for the membrane localization and function of these proteins.Prenylated proteins share characteristic C-terminal sequences includingCAAX (C, Cys; A, an aliphatic amino acid; X, another amino acid), XXCC,or XCXC. Three post-translational processing steps have been describedfor proteins having a C-terminal CAAX sequence: addition of either a 15carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoid to the Cysresidue, proteolytic cleavage of the last 3 amino acids, and methylationof the new C-terminal carboxylate (Cox, A. D. and Der, C. J. (1992a).Critical Rev. Oncogenesis 3:365-400; Newman, C. M. H. and Magee, A. I.(1993). Biochim. Biophys. Acta 1155:79-96). Some proteins may also havea fourth modification: palmitoylation of one or two Cys residuesN-terminal to the farnesylated Cys. While some mammalian cell proteinsterminating in XCXC are carboxymethylated, it is not clear whethercarboxy methylation follows prenylation of proteins terminating with aXXCC motif (Clarke, S. (1992). Annu. Rev. Biochem. 61, 355-386).

For all of the prenylated proteins, addition of the isoprenoid is thefirst step and is required for the subsequent steps (Cox, A D. and Der,C. J. (1992a). Critical Rev. Oncogenesis 3:365-400; Cox, A. D. and Der,C. J. (1992b) Current Opinion Cell Biol. 4:1008-1016).

Three enzymes have been described that catalyze protein prenylation:farnesyl-protein transferase (FPTase), geranylgeranyl-proteintransferase type I (GGPTase-I), and geranylgeranyl-protein transferasetype-II (GGPTase-II, also called Rab GGPTase). These enzymes are foundin both yeast and mammalian cells (Clarke, 1992;

Schafer, W. R. and Rine, J. (1992) Annu. Rev. Genet. 30:209-237). Eachof these enzymes selectively uses farnesyl diphosphate orgeranyl-geranyl diphosphate as the isoprenoid donor and selectivelyrecognizes the protein substrate. FPTase farnesylates CaaX-containingproteins that end with Ser, Met, Cys, Gln or Ala. For FPTase, CaaXtetrapeptides comprise the minimum region required for interaction ofthe protein substrate with the enzyme. The enzymologicalcharacterization of these three enzymes has demonstrated that it ispossible to selectively inhibit one with little inhibitory effect on theothers (Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara,M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J.B., J. Biol. Chem., 266:17438 (1991), U.S. Pat. No. 5,470,832).

The prenylation reactions have been shown genetically to be essentialfor the function of a variety of proteins (Clarke, 1992; Cox and Der,1992a; Gibbs, J. B. (1991). Cell 65: 1-4; Newman and Magee, 1993;Schafer and Rine, 1992). This requirement often is demonstrated bymutating the CaaX Cys acceptors so that the proteins can no longer beprenylated. The resulting proteins are devoid of their centralbiological activity. These studies provide a genetic “proof ofprinciple” indicating that inhibitors of prenylation can alter thephysiological responses regulated by prenylated proteins.

The Ras protein is part of a signaling pathway that links cell surfacegrowth factor receptors to nuclear signals initiating cellularproliferation. Biological and biochemical studies of Ras action indicatethat Ras functions like a G-regulatory protein. In the inactive state,Ras is bound to GDP. Upon growth factor receptor activation, Ras isinduced to exchange GDP for GTP and undergoes a conformational change.The GTP-bound form of Ras propagates the growth stimulatory signal untilthe signal is terminated by the intrinsic GTPase activity of Ras, whichreturns the protein to its inactive GDP bound form (D. R. Lowy and D. M.Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)). Activation of Rasleads to activation of multiple intracellular signal transductionpathways, including the MAP Kinase pathway and the Rho/Rac pathway(Joneson et al., Science 271:810-812).

Mutated ras genes are found in many human cancers, including colorectalcarcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Theprotein products of these genes are defective in their GTPase activityand constitutively transmit a growth stimulatory signal.

The Ras protein is one of several proteins that are known to undergopost-translational modification. Farnesyl-protein transferase utilizesfarnesyl pyrophosphate to covalently modify the Cys thiol group of theRas CAAX box with a farnesyl group (Reiss et al., Cell, 62:81-88 (1990);Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al.,Science, 249:1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA,87:7541-7545 (1990)).

Ras must be localized to the plasma membrane for both normal andoncogenic functions. At least 3 post-translational modifications areinvolved with Ras membrane localization, and all 3 modifications occurat the C-terminus of Ras. The Ras C-terminus contains a sequence motiftermed a “CAAX” or “Cys—Aaa¹—Aaa²—Xaa” box (Cys is cysteine, Aaa is analiphatic amino acid, the Xaa is any amino acid) (Willumsen et al.,Nature 310:583-586 (1984)). Depending on the specific sequence, thismotif serves as a signal sequence for the enzymes farnesyl-proteintransferase or geranylgeranyl-protein transferase, which catalyze thealkylation of the cysteine residue of the CAAX motif with a C₁₅ or C₂₀isoprenoid, respectively. (S. Clarke., Ann. Rev. Biochem. 61:355-386(1992); W. R. Schafer and J. Rine, Ann. Rev Genetics 30:209-237 (1992)).Direct inhibition of farnesyl-protein transferase would be more specificand attended by fewer side effects than would occur with the requireddose of a general inhibitor of isoprene biosynthesis.

Other farnesylated proteins include the Ras-related GTP-binding proteinssuch as RhoB, fungal mating factors, the nuclear lamins, and the gammasubunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994)have identified a peroxisome associated protein Pxf which is alsofarnesylated. James, et al., have also suggested that there arefarnesylated proteins of unknown structure and function in addition tothose listed above.

Inhibitors of farnesyl-protein transferase (FPTase) have been describedin two general classes. The first class includes analogs of farnesyldiphosphate (FPP), while the second is related to protein :substrates(e.g., Ras) for the enzyme. The peptide derived inhibitors that havebeen described are generally cysteine containing molecules that arerelated to the CAAX motif that is the signal for protein prenylation.(Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS,88:732-736 (1991)). Such inhibitors may inhibit protein prenylationwhile serving as alternate substrates for the farnesyl-proteintransferase enzyme, or may be purely competitive inhibitors (U.S. Pat.No. 5,141,851, University of Texas; N. E. Kohl et al., Science,260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).

Mammalian cells express four types of Ras proteins (H-, N-, K4A-, andK4B-Ras) among which K4B-Ras is the most frequently mutated form of Rasin human cancers. The genes that encode these proteins are abbreviatedH-ras, N-ras, K4A-ras and K4B-ras respectively.

H-ras is an abbreviation for Harvey-ras. K4A-ras and K4B-ras areabbreviations for the Kirsten splice variants of ras that contain the 4Aand 4B exons, respectively. Inhibition of farnesyl-protein transferasehas been shown to block the growth of H-ras-transformed cells in softagar and to modify other aspects of their transformed phenotype. It hasalso been demonstrated that certain inhibitors of farnesyl-proteintransferase selectively block the processing of the H-Ras oncoproteinintracellularly (N. E. Kohl et al., Science, 260:1934-1937 (1993) and G.L. James et al., Science, 260:1937-1942 (1993). Recently, it has beenshown that an inhibitor of farnesyl-protein transferase blocks thegrowth of H-ras-dependent tumors in nude mice (N. E. Kohl et al., Proc.Natl. Acad. Sci U.S.A., 91:9141-9145 (1994) and induces regression ofmammary and salivary carcinomas in H-ras transgenic mice (N. E. Kohl etal., Nature Medicine, 1:792-797 (1995).

Indirect inhibition of farnesyl-protein transferase in vivo has beendemonstrated with lovastatin Merck & Co., Rahway, N.J.) and compactin(Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science245:379 (1989)). These drugs inhibit HMG-CoA reductase, the ratelimiting enzyme for the production of polyisoprenoids including farnesylpyrophosphate. Inhibition of farnesyl pyrophosphate biosynthesis byinhibiting HMG-CoA reductase blocks Ras membrane localization in:cultured cells.

It has been disclosed that the lysine-rich region and terminal CVIMsequence of the C-terminus of K-RasB confer resistance to inhibition ofthe cellular processing of that protein by certain selective FPTaseinhibitors. James, et al., J. Biol. Chem. 270, 6221 (1995) Those FPTaseinhibitors were effective in inhibiting the processing of H-Rasproteins. James et al. suggested that prenylation of the K4B-Ras proteinby GGTase-I contributed to the resistance to the selective FPTaseinhibitors.

Selective inhibitors of GGTase-I have been previously disclosed (see forexample U.S. Pat. No. 5,470,832, issued November 28, 1995). Othercompounds have been described as selective inhibitors of GGTase-I (seefor example PCT Publication No. WO 96/21456). Combinations of aselective inhibitor of FPTase and a selective inhibitor of GGTase-I havebeen disclosed as useful in the treatment of cancer (PCT Publication No.WO 97/34664).

Several groups of scientists have recently disclosed compounds that arenon-selective FPTase/GGTase-I inhibitors. (Nagasu et al. CancerResearch, 55:5310-5314 (1995); PCT application WO 95/25086).

It is the object of the instant invention to provide a prenyl-proteintransferase inhibitor which is efficacious in vivo as an inhibitor ofgeranylgeranyl-protein transferase type I (GGTase-I), also known as CAAXGGTase.

It is also the object of the present invention to provide a compoundwhich inhibits the cellular processing of both the H-Ras protein and theK4B-Ras protein.

It is also the object of the present invention to provide a compoundwhich is efficacious in vivo as an inhibitor of the growth of cancercells characterized by a mutated K4B-Ras protein.

A composition which comprises such an inhibitor compound is used in thepresent invention to treat cancer.

SUMMARY OF THE INVENTION

The present invention comprises piperazine-containing compounds whichinhibit prenyl-protein transferases. Further contained in this inventionare chemotherapeutic compositions containing these prenyl transferaseinhibitors and methods for their production.

The compounds of this invention are illustrated by the formula A:

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful in the inhibition ofprenyl-protein transferases and the prenylation of the oncogene proteinRas. In a first embodiment of this invention, the inhibitors ofprenyl-protein transferases are illustrated by the formula A:

wherein:

R^(1a) and R^(1b) are independently selected from:

a) hydrogen,

b) aryl, heterocycle, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,R¹⁰O—, R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from unsubstituted orsubstituted aryl, heterocyclic, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, R¹⁰O—, R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—,CN, R¹⁰OC(O)—, N₃, —N(R¹⁰)₂, and R¹¹OC(O)— NR¹⁰—;

R^(1c) is independently selected from:

a) hydrogen,

b) unsubstituted or substituted aryl, unsubstituted or substitutedheterocycle, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, R¹⁰O—,R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from unsubstituted orsubstituted aryl, unsubstituted or substituted heterocycle, C₃-C₁₀cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, R¹⁰O—, R¹¹S(O)m—,R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂and R¹¹OC(O)—NR¹⁰—;or two R^(1c)s on the same carbon are combined withthat carbon to form a C₄-C₆ cycloalkyl;

R² and R³ are independently selected from: H; unsubstituted orsubstituted C₁₋₈ alkyl, unsubstituted or substituted C₂₋₈ alkenyl,unsubstituted or substituted C₂₋₈ alkynyl, unsubstituted or substitutedaryl, unsubstituted or substituted heterocycle,

 wherein the substituted group is substituted with one or more of:

R² and R³ are attached to the same C atom and are combined to form—(CH₂)u— wherein one of the carbon atoms is optionally replaced by amoiety selected from: O, S(O)m, —NC(O)—, and —N(COR¹⁰)—;

R⁴ is selected from H and CH₃; and any two of R², R³ and R⁴ areoptionally attached to the same carbon atom;

R⁶, R⁷ and R^(7a) are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, heterocycle, aryl, aroyl, heteroaroyl, arylsulfonyl,heteroarylsulfonyl, unsubstituted or substituted with:

R⁶ and R⁷ may be joined in a ring;

R⁷ and R^(7a) may be joined in a ring;

R⁶a is selected from: C₁₋₄ alkyl, C₃₋₆ cycloalkyl, heterocycle, aryl,unsubstituted or substituted with:

R⁸ is independently selected from:

a) hydrogen,

b) unsubstituted or substituted aryl, unsubstituted or substitutedheterocycle, unsubstituted or substituted C₃-C₁₀ cycloalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, perfluoroalkyl, F, Cl, Br, R¹⁰O—, R¹¹S(O)m—,R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃,—N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, cyanophenyl,heterocycle, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,perfluoroalkyl, F, Cl, Br, R¹⁰O—, R¹¹S(O)m—, R¹⁰C(O)NH—, (R¹⁰)₂NC(O)—,R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰ ₂, or R¹⁰OC(O)NH—;

R⁹ is selected from:

a) hydrogen,

b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, R¹⁰O—, R¹lS(O)m—,R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃,—N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl unsubstituted or substituted by perfluoroalkyl, F, Cl,Br, R¹⁰O—, R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN,R¹⁰C(O)—, N₃, —N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ and A² are independently selected from: a bond, —CH═CH—, —C═C—,—C(O)—, —C(O)NR¹⁰—, —NR¹⁰C(O)—, O, —N(R¹⁰)—, —S(O)₂N(R¹⁰)—,—N(R¹⁰)S(O)₂—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰—, —C(O)O—, S(O)m or a bond;

A⁴ is selected from: bond, —O—and —NR¹⁰—;

G is H₂ or O, provided that G is not H₂, if A³ is a bond;

V is selected from:

a) hydrogen,

b) heterocycle,

c) aryl,

d) C₃-C₂₀ alkyl wherein from 0 to 4 carbon atoms are replaced with aheteroatom selected from O, S, and N, and

e) C₂-C₂₀ alkenyl, provided that V is not hydrogen if A¹ is S(O)m and Vis not hydrogen if Al is a bond, n is 0 and A² is S(O)m;

W is a heterocycle;

Z is an unsubstituted or substituted multicyclic ring, wherein thesubstituted multicyclic ring is substituted with one or two moietiesselected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

m is 0, 1 or 2;

n is 0, 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

q is 1 or 2;

r is 0 to 5, provided that r is 0 when V is hydrogen;

s is 0 or 1;

t is 0 or 1;

u is 4 or 5; and

v is 0, 1, 2 or 3;

or the pharmaceutically acceptable salts thereof

In a preferred embodiment of this invention, the inhibitors ofprenyl-protein transferase are illustrated by the formula A:

wherein:

R^(1a) is independently selected from: hydrogen or C₁-C₆ alkyl;

R^(1b) is independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from unsubstituted orsubstituted aryl, heterocycle, cycloalkyl, alkenyl, R¹⁰O— and —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;or two R^(1c)s on the same carbon are combined with thatcarbon to form a C₄-C₆ cycloalkyl;

R³ and R⁴ are independently selected from H and CH₃;

R² is H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substitutedwith one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

5)

 and any two of R², R³, R⁴, and R⁵ are optionally attached to the samecarbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—;

R⁹ is selected from:

a) hydrogen,

b) C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F, Cl, R¹⁰O—,R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂,or R¹⁰C(O)NR¹⁰—, and

c) C₁-C₆ alkyl unsubstituted or substituted by C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹¹S(O)m—, R¹⁰C(O)NR¹⁰—, CN, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—,—N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ and A² are independently selected from: a bond, —CH═CH—, —C≡C—,—C(O)—, —C(O)NR¹⁰—, O, —N(R¹⁰)—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰—, —C(O)O—, S(O)m or a bond;

A⁴ is selected from: bond, —O—and —NR¹⁰—;

G is H₂ or O, provided that G is not H₂, if A³ is a bond;

V is selected from:

a) hydrogen,

b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,isoquinolinyl, and thienyl,

c) aryl,

d) C₁-C₂₀ alkyl wherein from 0 to 4 carbon atoms are replaced with a aheteroatom selected from O, S, and N, and

e) C₂-C₂₀ alkenyl, and provided that V is not hydrogen if A¹ is S(O)mand V is not hydrogen if Al is a bond, n is 0 and A² is S(O)m;

W is a heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, orisoquinolinyl;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

m is 0, 1 or 2;

n is 0, 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

r is 0 to 5, provided that r is 0 when V is hydrogen;

s is 0 or 1;

t is 0 or 1; and

v is 0, 1, 2 or 3; provided that v is not 0 if A³ is a bond;

or the pharmaceutically acceptable salts thereof.

A preferred embodiment of the compounds of this invention areillustrated by the formula B:

wherein:

R^(1a) and R^(1b) are independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;or two R^(1c)s on the same carbon are combined with thatcarbon to form a C₄-C₆ cycloalkyl;

R³ is selected from H and CH₃;

R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substitutedwith one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl,unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ and A² are independently selected from: a bond, —CH═CH—, —C≡C—,—C(O)—, —C(O)NR¹⁰—, O, —N(R¹⁰)—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰—, —C(O)O— or S(O)m;

A⁴ is selected from: bond, —O—and —NR¹⁰—;

V is selected from:

a) hydrogen,

b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,isoquinolinyl, and thienyl,

c) aryl,

d) C₁-C₂₀ alkyl wherein from 0 to 4 carbon atoms are replaced with a aheteroatom selected from O, S, and N, and

e) C₂-C₂₀ alkenyl, and provided that V is not hydrogen if A¹ is S(O)mand V is not hydrogen if A¹ is a bond, n is 0 and A² is S(O)m;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

m is 0, 1 or 2;

n is 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

r is 0 to 5, provided that r is 0 when V is hydrogen; and

v is 0, 1, 2 or 3; provided that v is not 0 if A³ is a bond;

or the pharmaceutically acceptable salts thereof.

Another preferred embodiment of the compounds of this invention areillustrated by the formula C:

wherein:

R^(1a) and R^(1b) are independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(RI⁰)₂ andR¹¹OC(O)—NR¹⁰—;or two R^(1c)s on the same carbon are combined with thatcarbon to form a C₄-C₆ cycloalkyl;

R³ is selected from H and CH₃;

R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substitutedwith one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

⁵)

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ and A² are independently selected from: a bond, —CH═CH—, —C≡C—,—C(O)—, —C(O)NR¹⁰—, O, —N(R¹⁰)—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰—, —C(O)O— or S(O)m;

A⁴ is selected from: bond, —O—and —NR¹⁰—;

V is selected from:

a) hydrogen,

b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl,thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl,isoquinolinyl, and thienyl,

c) aryl,

d) C₁-C₂₀ alkyl wherein from 0 to 4 carbon atoms are replaced with a aheteroatom selected from O, S, and N, and

e) C₂-C₂₀ alkenyl, and provided that V is not hydrogen if A¹ is S(O)mand V is not hydrogen if Al is a bond, n is 0 and A² is S(O)m;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

 m is 0, 1 or 2;

n is 0, 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

r is 0 to 5, provided that r is 0 when V is hydrogen; and

v is 0, 1, 2 or 3; provided that v is not 0 if A³ is a bond;

or the pharmaceutically acceptable salts thereof.

A further embodiment of the compounds of this invention is illustratedby the formula D:

wherein:

R^(1a) and R^(1b) are independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;

R³ is selected from H and CH₃;

R² is selected from H; O or C₁₋₅ alkyl, unbranched or branched,unsubstituted or substituted with one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

5)

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰ ₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ is selected from: a bond, —CH═CH—, —C═C—, —C(O)—, —C(O)NR¹⁰—, O,—N(R¹⁰)—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—;

A⁴ is selected from: bond and —O—;

V is selected from:

a) heterocycle selected from pyridinyl and quinolinyl, and

b) aryl;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁-₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

m is 0, 1 or 2;

n is 0, 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

r is 0 to 5, and

v is 0, 1, 2 or 3;

or the pharmaceutically acceptable salts thereof.

Another embodiment of the compounds of this invention is illustrated bythe formula E:

wherein:

R^(1a) and R^(1b) are independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;

R³ is selected from H and CH₃;

R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substitutedwith one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

5)

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄alkoxy,

b) halogen, or

c) aryl or heterocycle;

R_(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A¹ is selected from: a bond, —CH═CH—, —C≡C—, —C(O)—, —C(O)NR¹⁰—, O,—N(R¹⁰)—, or S(O)m;

A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—;

A⁴ is selected from: bond and —O—;

V is selected from:

a) heterocycle selected from pyridinyl and quinolinyl, and

b) aryl;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

 

m is 0, 1 or 2;

n is 0, 1, 2, 3 or 4;

p is 0, 1, 2, 3 or 4;

r is 0 to 5, and

v is 0, 1, 2 or 3;

or the pharmaceutically acceptable salts thereof.

A still further embodiment of the compounds of this invention isillustrated by the formula F:

wherein:

R^(1b) is independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;

R³ is selected from H and CH₃;

R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or su tituted withone or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

5)

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—;

A⁴ is selected from: bond and —O—;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

m is 0, 1 or 2;

p is 1, 2 or 3;

r is 0 to 5, and

v is 0, 1, 2 or 3;

or the pharmaceutically acceptable salts thereof

Another further embodiment of the compounds of this invention isillustrated by the formula G:

wherein:

R^(1b) is independently selected from:

a) hydrogen,

b) aryl, heterocycle, cycloalkyl, R¹⁰O—, —N(R¹⁰)₂ or C₂-C₆ alkenyl,

c) C₁-C₆ alkyl unsubstituted or substituted by aryl, heterocycle,cycloalkyl, alkenyl, R¹⁰O—, or —N(R¹⁰)₂;

R^(1c) is independently selected from:

a) hydrogen,

b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂,R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—,

c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent onthe substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—,(R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ andR¹¹OC(O)—NR¹⁰—;

R³ is selected from H and CH₃;

R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substitutedwith one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^(6a), SO₂R^(6a), or

5)

 and R² and R³ are optionally attached to the same carbon atom;

R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆cycloalkyl, aryl, heterocycle, unsubstituted or substituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted orsubstituted with:

a) C₁₋₄ alkoxy,

b) halogen, or

c) aryl or heterocycle;

R⁸ is independently selected from:

a) hydrogen,

b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F,Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂,or R¹¹OC(O)NR¹⁰—, and

c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, RI°C(O)NR¹⁰—, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—;

R^(9a) is hydrogen or methyl;

R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl andaryl;

R¹¹ is independently selected from C₁-C₆ alkyl and aryl;

A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—;

A⁴ is selected from: bond and —O—;

Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring,wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substitutedwith one or two moieties selected from the following:

a) C₁₋₄ alkoxy,

b) NR⁶R⁷,

c) C₃₋₆ cycloalkyl,

d) —NR⁶C(O)R⁷,

e) HO,

f) —S(O)mR^(6a),

g) halogen,

h) perfluoroalkyl, and

i) C₁₋₄ alkyl;

C₇-C₁₀ multicyclic alkyl ring is selected from:

m is 0, 1 or 2;

p is 1, 2 or 3;

r is 0 to 5, and

v is 0, 1, 2 or 3;

or the pharmaceutically acceptable salts thereof

Specific examples of compounds of this invention are as follows:

1-(4-Cyanobenzyl)-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

R/S1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-(4-Cyanobenzyl)-5-[1-(2-acetyloxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]piperazine-4-(N-1-adamantyl)carboxamide

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(N-1-adamantyl)carbonylpiperazine

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-[N-(1R)-(−)-10-camphorsulfonyl]piperazine

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(1-adamantylmethyl)piperazine

1-(4-Cyanobenzyl)-5-[1-(2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-(4-Cyanobenzyl)-5-[1-(1-(adamant-1-yl)methyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl] piperazine-4-carboxylic acid(2-norbornane)methyl ester

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-piperazine-4-carboxylic acid(2-norbornane)methyl ester

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(2-bicyclo-[2.2.2]-octylcarbonyl)piperazine

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(2-norbornanecarbonyl)piperazine

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-cis/trans-(2,6,6-trimethylbicyclo[3.1.1]heptanecarbonyl)-piperazine

1-(4′-Cyanobenzyl)-2-methyl-imidazol-5-ylmethylpiperazine-4-(N-1-adamantyl)carboxamide

or the pharmaceutically acceptable salts or optical isomers thereof.

The compounds of the instant invention differ from previously disclosedpiperazinone-containing and piperazine-containing compounds, (PCT Publ.No. WO 96/30343—Oct. 3, 1996; U.S. Pat. No. 5,856,326—Jan. 5, 1999; PCTPubl. No. WO 96/31501—Oct. 10, 1996; PCT Publ. No. WO 97/36593—Oct. 9,1997; PCT Publ. No. WO 97/36592—Oct. 9, 1997) that were described asinhibitors of farnesyl-protein transferase (FPTase), in that, amongother things, the instant compounds are dual inhibitors offarnesyl-protein transferase and geranylgeranyl-protein transferase typeI (GGTase-I).

In one embodiment of the invention, the compounds are furthercharacterized in that the inhibitory activity of the compounds againstGGTase-I is greater than the inhibitory activity against FPTase.Preferably, the compounds of this embodiment of the instant inventioninhibit FPTase in vitro Example 16) at an IC₅₀ of less than 1 μM andinhibit GGTase-I in vitro (Example 17) at an IC₅₀ of less than 50 nM.Also preferably, the compounds of this embodiment of the instantinvention inhibit the cellular processing of the Rap 1 protein (Example22, Protocol C) at an EC₅₀ of less than about 1 μM. More preferably, thecompounds of this embodiment of the instant invention inhibit thecellular processing of the Rapl protein (Example 22, Protocol C) at anEC₅₀ of less than about 50 nM. Also more preferably, the ratio of theIC₅₀ of the compounds of this embodiment of the instant invention for invitro inhibition of FPTase to the IC₅₀ of the compounds of the instantinvention for in vitro inhibition of GGTase type I is greater than 25.Also more preferably, the ratio of the EC₅₀ of the compounds of thisembodiment of the instant invention for inhibition of the cellularprocessing of the hDJ protein (Example 21) to the EC₅₀ of the compoundsof the instant invention for inhibition of the cellular processing ofthe Rap 1 protein is about equal to or less than 1.

In a second embodiment of the compounds of the instant invention, thecompounds are further characterized in that the inhibitory activity ofthe compounds against FPTase is greater than the inhibitory activityagainst GGTase-I. Preferably, the compounds of this second embodiment ofthe instant invention inhibit FPTase in vitro (Example 16) at an IC₅₀ ofless than 100 nM and inhibit GGTase-I in vitro (Example 17) at an IC₅₀of less than 5 μM. Preferably, the compounds of this second embodimentof the instant invention inhibit the cellular processing of the hDJprotein (Example 21) at an EC₅₀ of less than about 250 nM. Alsopreferably, the compounds of this second embodiment of the instantinvention inhibit the cellular processing of the Rap 1 protein (Example22, Protocol C) at an EC₅₀ of less than about 10 μM. More preferably,the compounds of this second embodiment of the instant invention inhibitthe cellular processing of the Rap 1 protein (Example 22, Protocol C) atan EC₅₀ of less than about 1 μM. Also more preferably, the ratio of theIC₅₀ of the compounds of this embodiment of the instant invention for invitro inhibition of GGTase type I to the IC₅₀ of the compounds of theinstant invention for in vitro inhibition of FPTase is greater than 1and less than 25. Also more preferably, the ratio of the EC₅₀ of thecompounds of this second embodiment of the instant invention forinhibition of the cellular processing of the hDJ protein (Example 21) tothe EC₅₀ of the compounds of the instant invention for inhibition of thecellular processing of the Rap 1 protein is between about 1 and about100.

The compounds of the present invention may have asymmetric centers andoccur as racemates, racemic mixtures, and as individual diastereomers,with all possible isomers, including optical isomers, being included inthe present invention. When any variable (e.g. aryl, heterocycle, R¹, R²etc.) occurs more than one time in any constituent, its definition oneach occurrence is independent at every other occurrence. Also,combinations of substituents/or variables are permissible only if suchcombinations result in stable compounds.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms; “alkoxy” represents an alkyl group ofindicated number of carbon atoms attached through an oxygen bridge.“Halogen” or “halo” as used herein means fluoro, chloro, bromo and iodo.

As used herein, “cycloalkyl” is intended to include monocyclic saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms. Examples of such cycloalkyl groups includes, but are not limitedto, cyclopropyl, cyclobutyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 members in each ring, wherein at leastone ring is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl and biphenyl.

The term heterocycle or heterocyclic, as used herein, represents astable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclicheterocyclic ring which is either saturated or unsaturated, and whichconsists of carbon atoms and from one to four heteroatoms selected fromthe group consisting of N, O, and S, and including any bicyclic group inwhich any of the above-defined heterocyclic rings is fused to a benzenering. The term heterocycle or heterocyclic, as used herein, includesheteroaryl moieties. The heterocyclic ring may be attached at anyheteroatom or carbon atom which results in the creation of a stablestructure. Examples of such heterocyclic elements include, but are notlimited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl,benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl,benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranylsulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl,indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl,isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl,2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl,pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl,pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl,tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl,thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl,thienothienyl, and thienyl.

As used herein, “heteroaryl” is intended to mean any stable monocyclicor bicyclic carbon ring of up to 7 members in each ring, wherein atleast one ring is aromatic and wherein from one to four carbon atoms arereplaced by heteroatoms selected from the group consisting of N, O, andS. Examples of such heterocyclic elements include, but are not limitedto, benzimid-azolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl,benzo-thiopyranyl, benzofuryl, benzothiazolyl, benzothienyl,benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydro-benzothiopyranylsulfone, furyl, imidazolyl, indolinyl, indolyl, isochromanyl,isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl,pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl,quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, thiazolyl, thienofuryl, thienothienyl, andthienyl.

As used herein in the definition of R² and R³, the term “the substitutedgroup” intended to mean a substituted C₁₋₈ alkyl, substituted C₂₋₈alkenyl, substituted C₂₋₈ alkynyl, substituted aryl or substitutedheterocycle from which the substituent(s) R² and R³ are selected.

As used herein in the definition of R⁶, R^(6a), R⁷ and R^(7a), thesubstituted C₁₋₈ alkyl, substituted C₃₋₆ cycloalkyl, substituted aroyl,substituted aryl, substituted heteroaroyl, substituted arylsulfonyl,substituted heteroarylsulfonyl and substituted heterocycle includemoieties containing from 1 to 3 substituents in addition to the point ofattachment to the rest of the compound. Preferably, such substituentsare selected from the group which includes but is not limited to F, Cl,Br, CF₃, NH₂, N(C₁-C₆ alkyl₂, NO₂, CN, (C₁-C₆ alkyl)O—, —OH, (C₁-C₆alkyl)S(O)m—, (C₁-C₆ alkyl)C(O)NH—, H₂N—C(NH)—, (C₁-C₆ alkyl)C(O)—,(C₁-C₆ alkyl)OC(O)—, N₃, (C₁-C₆ alkyl)OC(O)NH—, phenyl, pyridyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl,isothiazolyl and C₁-C₂₀ alkyl.

As used herein, “multicyclic ring” is intended to include polycyclicsaturated and unsaturated aliphatic hydrocarbon groups which optionallycontain 1-3 heteroatoms, selected from N, O and S. Examples of suchmulticyclic rings includes, but are not limited to:

As used herein, “multicyclic alkyl ring” is intended to includepolycyclic saturated and unsaturated aliphatic hydrocarbon groups havingthe specified number of carbon atoms. Examples of such cycloalkyl groupsincludes, but are not limited to:

When R² and R³ are combined to form—(CH₂)u—, and when two R^(1c) son thesame carbon are combined with that carbon to form a C₄-C₆ cycloalkyl,cyclic moieties are formed. Examples of such cyclic moieties include,but are not limited to:

In addition, with respect to R² and R³, such cyclic moieties mayoptionally include a heteroatom(s). Examples of suchheteroatom-containing cyclic moieties include, but are not limited to:

The moiety formed when, in the definition of R⁶, R⁷ and R^(7a), R⁶ andR⁷ or R⁷ and R^(7a) are joined to form a ring, is illustrated by, butnot limited to, the following:

Lines drawn into the ring systems from substituents (such as from R²,R³, R⁴ etc.) represent that the indicated bond may be attached to any ofthe substitutable ring carbon atoms.

Preferably, R^(1a) and R^(1b) are independently selected from: hydrogen,—N(R¹⁰ ₂, R¹⁰C(O)NR¹⁰— or unsubstituted or substituted C₁-C₆ alkylwherein the substituent on the substituted C₁-C₆ alkyl is selected fromunsubstituted or substituted phenyl, —N(R¹⁰ ₂, R¹⁰O— and R¹⁰C(O)NR¹⁰—.

Preferably, R² is selected from: hydrogen,

and an unsubstituted or substituted group, the group selected from C₁₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈ alkynyl; wherein the substituted group issubstituted with one or more of:

1) aryl or heterocycle,

2) OR⁶, and

3) SR^(6a), SO₂R^(6a).

Preferably, R³ is selected from hydrogen and methyl.

Preferably, R⁴ is hydrogen.

Preferably, R⁶ and R⁷ are selected from: hydrogen, unsubstituted orsubstituted C₁-C₆ alkyl, unsubstituted or substituted aryl andunsubstituted or substituted C₃-C₆ cycloalkyl.

Preferably, R^(6a) is unsubstituted or substituted C₁-C₆.

Preferably, R⁹ is hydrogen or methyl.

Preferably, R¹⁰ is selected from H, C₁-C₆ alkyl and benzyl.

Preferably, A¹ and A² are independently selected from: a bond,—C(O)NR¹⁰—, —NR¹⁰C(O)—, O, —N(R¹⁰)—, —S(O)₂N(R¹⁰)— and —N(R¹⁰)S(O)₂—.Most preferably, A¹ and A² are a bond.

Preferably, A³ is selected from: —C(O)—, —C(O)NR¹⁰— and —C(O)O—.

Preferably, A⁴ is a bond.

Preferably, V is selected from heteroaryl and aryl. More preferably, Vis phenyl.

Preferably, W is selected from imidazolyl, pyridinyl, thiazolyl,indolyl, quinolinyl, or isoquinolinyl. More preferably, W is imidazolyland pyridyl.

Preferably, Z is a unsubstituted or substituted C₇-C₁₀ multicyclic alkylring.

Preferably, the C₇-C₁₀ multicyclic alkyl ring is selected from:

Preferably, n and r are independently 0, 1, or 2.

Preferably p is 1, 2 or 3.

Preferably s is 0.

Preferably t is 1.

Preferably v is 0, 1 or 2.

Preferably, the moiety

is selected from:

Preferably, the moiety A¹ (CR^(1a) ₂)_(n)A²(CR^(1a) ₂)n is not a bond.

It is intended that the definition of any substituent or variable (e.g.,R^(1a), R⁹, n, etc.) at a particular location in a molecule beindependent of its definitions elsewhere in that molecule. Thus,—N(R¹⁰)₂represents —NHH, —NHCH₃, —NHC₂H₅, etc. It is understood thatsubstituents and substitution patterns on the compounds of the instantinvention can be selected by one of ordinary skill in the art to providecompounds that are chemically stable and that can be readily synthesizedby techniques known in the art, as well as those methods set forthbelow, from readily available starting materials.

The pharmaceutically acceptable salts of the compounds of this inventioninclude the conventional non-toxic salts of the compounds of thisinvention as formed, e.g., from non-toxic inorganic or organic acids.For example, such conventional non-toxic salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, nitric and the like: and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, trifluoroacetic and the like.

The pharmaceutically acceptable salts of the compounds of this inventioncan be synthesized from the compounds of this invention which contain abasic moiety by conventional chemical methods. Generally, the salts areprepared either by ion exchange chromatography or by reacting the freebase with stoichiometric amounts or with an excess of the desiredsalt-forming inorganic or organic acid in a suitable solvent or variouscombinations of solvents.

Reactions used to generate the compounds of this invention are preparedby employing reactions as shown in the Schemes 1-21, in addition toother standard manipulations such as ester hydrolysis, cleavage ofprotecting groups, etc., as may be known in the literature orexemplified in the experimental procedures. Substituents R, R^(a) andR^(b), as shown in the Schemes, represent the substituents R², R³ andR⁴; however their point of attachment to the ring is illustrative onlyand is not meant to be limiting. Substituent Z′, as shown in theSchemes, represents the substiutent Z as defined hereinabove or aprotected precursor thereof.

These reactions may be employed in a linear sequence to provide thecompounds of the invention or they may be used to synthesize fragmentswhich are subsequently joined by the alkylation reactions described inthe Schemes.

Synopsis of Schemes 1-21:

The requisite intermediates are in some cases commercially available, orcan be prepared according to literature procedures, for the most part.In Scheme 1, for example, the synthesis of 2-cycloalkylalkanoylsubstituted piperazines is outlined. Boc-protected amino acids I,available commercially or by procedures known to those skilled in theart, can be coupled to N-benzyl amino acid esters using a variety ofdehydrating agents such as DCC (dicyclohexycarbodiimide) or EDC.HCl(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) in asolvent such as methylene chloride, chloroform, dichloroethane, ordimethylformamide. The product II is then deprotected with acid, forexample hydrogen chloride in chloroform or ethyl acetate, ortrifluoroacetic acid in methylene chloride, and cyclized under weaklybasic conditions to give the diketopiperazine III. Reduction of III withlithium aluminum hydride in refluxing ether gives the piperazine IV,which is protected as the Boc derivative V. The N-benzyl group can becleaved under standard conditions of hydrogenation, e.g., 10% palladiumon carbon at 60 psi hydrogen on a Parr apparatus for 24-48 h. Theproduct VI can be reacted with a suitably substituted carboxylic acid toprovide the piperazine VII; a final acid deprotection as previouslydescribed gives the intermediate VIII (Scheme 2). The intermediate VIIIcan itself be reductively alkylated with a variety of aldehydes, such asIX. The aldehydes can be prepared by standard procedures, such as thatdescribed by O. P. Goel, U. Krolls, M. Stier and S. Kesten in OrganicSyntheses, 1988, 67, 69-75, from the appropriate amino acid (Scheme 3).The reductive alkylation can be accomplished at pH 5-7 with a variety ofreducing agents, such as sodium triacetoxyborohydride or sodiumcyanoborohydride in a solvent such as dichloroethane, methanol ordimethylformamide. The product X can be deprotected to give the finalcompounds XI with trifluoroacetic acid in methylene chloride. The finalproduct XI is isolated in the salt form, for example, as atrifluoroacetate, hydrochloride or acetate salt, among others. Theproduct diamine XI can further be selectively protected to obtain XII,which can subsequently be reductively alkylated with a second aldehydeto obtain XIII. Removal of the protecting group, and conversion tocyclized products such as the dihydroimidazole XV can be accomplished byliterature procedures.

As shown in Scheme 4, the piperazine intermediate VIII can bereductively alkylated with other aldehydes such as1-trityl-4-imidazolyl-carboxaldehyde or1-trityl-4-imidazolyl-acetaldehyde, to give products such as XVI. Thetrityl protecting group can be removed from XVI to give XVII, oralternatively, XVI can first be treated with an alkyl halide thensubsequently deprotected to give the alkylated imidazole XVIII.Alternatively, the intermediate VIII can be acylated or sulfonylated bystandard techniques.

Incorporation of a hydroxyl moiety on the sidechain carbon alpha to theamide carbonyl of compounds of the formula XVIII can be accomplished asillustrated in Scheme 5. A suitably substituted primary alcohol XIXundergoes a one carbon homologation, via a Swern oxidation, nitrileaddition and hydrolysis, to provide the substituted hydroxyacetic acidXX. The trifluoromethyl ketal is formed and reacted with the previouslydescribed protected piperazine VI to provide, following deprotection,the intermediate XXI. Intermediate XXI can undergo a variety ofreactions at its unsubstituted nitrogen. For example, treatment of XXIwith a suitably substituted imidazolylmethyl halide to provide theinstant compound XXII.

Scheme 6 illustrates incorporation of a cycloalkylalkoxycarbonyl moietyonto the piperazine nitrogen. Thus a suitably substituted alcohol XXIIIis reacted with nitrophenylchloroformate to provide the intermediateXXIV, which is reacted with a suitably substituted piperazine to providethe instant compound XXV. An analogous reaction sequence alternativelyprovides the corresponding aminocarbonyl substitution on the piperazinenitrogen, as shown in Scheme 7.

Scheme 8 illustrates the preparation of compounds analogous to compoundXXXI wherein the alcohol utilized in the first step is a suitablysubstituted adamantanol. The scheme also illustrates the incorporationof an indole moiety for the substiutent W in place of the preferredbenzylimidazolyl moiety.

Scheme 9 illustrates synthesis of an instant compound wherein anon-hydrogen R^(9b) is incorporated in the instant compound. Thus, areadily available 4-substituted imidazole XXVI may be selectivelyiodinated to provide the 5-iodoimidazole XXVII. That imidazole may thenbe protected and coupled to a suitably substituted benzyl moiety toprovide intermediate XXVIII. Attachment of the imidazolyl nitrogen viaan ethyl linker to the piperazine nitrogen of intermediate XXI,described above, provides the instant compound XXIX.

Compounds of the instant invention wherein the A¹(CR^(1a) ₂)nA²(CR^(1a)₂)n linker is oxygen may be synthesized by methods known in the art, forexample as shown in Scheme 10. The suitably substituted phenol XXX maybe reacted with methyl N-(cyano)methanimidate to provide the4-phenoxy-imidazole XXXI. After selective protection of one of theimidazolyl nitrogens, the intermediate XXXII can undergo alkylationreactions as described for the benzylimidazoles in Scheme 9.

If the piperazine VIII is reductively alkylated with an aldehyde whichalso has a protected hydroxyl group, such as XXXIII in Scheme 11, theprotecting groups can be subsequently removed to unmask the hydroxylgroup The Boc protected amino alcohol XXXIV can then be utilized tosynthesize 2-aziridinyl-methylpiperazines such as XXXV.

5-Substituted piperazin-2-ones can be prepared as shown in Scheme 12.Reductive amination of Boc-protected amino aldehydes XXXVI (preparedfrom I as illustrated) gives rise to compound XXXVII. This is thenreacted with bromoacetyl bromide under Schotten-Baumann conditions; ringclosure is effected with a base such as sodium hydride in a polaraprotic solvent such as dimethylformamide to give XXXVIII. The carbamateprotecting group is removed under acidic conditions such astrifluoroacetic acid in methylene chloride, or hydrogen chloride gas inmethanol or ethyl acetate, and the resulting piperazine can then becarried on to final products as described in above.

The isomeric 3-substituted piperazin-2-ones can be prepared as describedin Scheme 13. The imine formed from arylcarboxamides IXL and2-aminoglycinal diethyl acetal (XL) can be reduced under a variety ofconditions, including sodium triacetoxyborohydride in dichloroethane, togive the amine XLI. A suitably substituted amino acid I can be coupledto amine XLI under standard conditions, and the resulting amide XLIIwhen treated with aqueous acid in tetrahydrofuran can cyclize to theunsaturated XLIII. Catalytic hydrogenation under standard conditionsgives the requisite intermediate XLIV, which is elaborated to finalproducts as described in above.

Reaction Scheme 14 provides an illustrative example of the synthesis ofcompounds of the instant invention wherein the substituents R² and R³are combined to form —(CH₂)u—. For example,1-aminocyclohexane-1-carboxylic acid XLV can be converted to thespiropiperazine XLVI essentially according to the procedures outlined inSchemes 1 and 2. The piperazine intermediate XLVI can be deprotected asbefore, and carried on to final products as described in Schemes 3-8. Itis understood that reagents utilized to provide the imidazolylalkylsubstituent may be readily replaced by other reagents well known in theart and readily available to provide other N-substituents on thepiperazine.

Scheme 15 illustrates the use of an optionally substituted homoserinelactone XLVII to prepare a Boc-protected piperazinone XLVIII.Intermediate XLVIII may be deprotected and reductively alkylated oracylated as illustrated in the previous Schemes. Alternatively, thehydroxyl moiety of intermediate XLVIII may be mesylated and displaced bya suitable nucleophile, such as the sodium salt of ethane thiol, toprovide an intermediate IL. Intermediate XLVIII may also be oxidized toprovide the carboxylic acid on intermediate L, which can be utilizedform an ester or amide moiety.

Amino acids of the general formula LI which have a sidechain not foundin natural amino acids may be prepared by the reactions illustrated inScheme 16 starting with the readily prepared imine LII.

Schemes 17-20 illustrate syntheses of suitably substituted aldehydesuseful in the syntheses of the instant compounds wherein the variable Wis present as a pyridyl moiety. Similar synthetic strategies forpreparing alkanols that incorporate other heterocyclic moieties forvariable W are also well known in the art. For example, Scheme 21illustrates the preparation of the corresponding quinoline aldehyde.Scheme 22 depicts a general method for synthesizing a key intermediateuseful in the preparation of a preferred embodiments of the instantinvention wherein V is phenyl and W is imidazole. A piperazine moietycan be readily added to this benzyl-imidazole intermediate as set forthin Scheme 23.

The instant compounds are useful as pharmaceutical agents for mammals,especially for humans. These compounds may be administered to patientsfor use in the treatment of cancer. Examples of the type of cancer whichmay be treated with the compounds of this invention include, but are notlimited to, colorectal carcinoma, exocrine pancreatic carcinoma, myeloidleukemias and neurological tumors. Such tumors may arise by mutations inthe ras genes themselves, mutations in the proteins that can regulateRas activity (i.e., neurofibromin (NF-1), neu, src, ab1, 1ck, fyn) or byother mechanisms.

The compounds of the instant invention inhibit prenyl-proteintransferase and the prenylation of the oncogene protein Ras. The instantcompounds may also inhibit tumor angiogenesis, thereby affecting thegrowth of tumors (J. Rak et al. Cancer Research, 55: 4575-4580 (1995)).Such anti-angiogenesis properties of the instant compounds may also beuseful in the treatment of certain forms of vision deficit related toretinal vascularization.

The compounds of this invention are also useful for inhibiting otherproliferative diseases, both benign and malignant, wherein Ras proteinsare aberrantly activated as a result of oncogenic mutation in othergenes (i.e., the Ras gene itself is not activated by mutation to anoncogenic form) with said inhibition being accomplished by theadministration of an effective amount of the compounds of the inventionto a mammal in need of such treatment. For example, a component of NF-1is a benign proliferative disorder.

The instant compounds may also be useful in the treatment of certainviral infections, in particular in the treatment of hepatitis delta andrelated viruses (J. S. Glenn et al. Science, 256:1331-1333 (1992).

The compounds of the instant invention are also useful in the preventionof restenosis after percutaneous transluminal coronary angioplasty byinhibiting neointimal formation (C. Indolfi et al. Nature medicine,1:541-545(1995).

The instant compounds may also be useful in the treatment and preventionof polycystic kidney disease (D. L. Schaffner et al. American Journal ofPathology, 142:1051-1060 (1993) and B. Cowley, Jr. et al. FASEB Journal,2:A3160 (1988)).

The instant compounds may also be useful for the treatment of fungalinfections.

The instant compounds may also be useful as inhibitors of proliferationof vascular smooth muscle cells and therefore useful in the preventionand therapy of arteriosclerosis and diabetic vascular pathologies.

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers, excipients or diluents, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including the intravenous, intramuscular, intraperitoneal, subcutaneous,rectal and topical routes of administration.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastro-intestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropylmethyl-cellulose orhydroxy-propylcellulose, or a time delay material such as ethylcellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyl-eneglycol or an oil medium, forexample peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxy-ethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of Formula A may also be administered in the form of asuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula A are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, sex and response of the individual patient, as well as theseverity of the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

The compounds of the instant invention may also be co-administered withother well known therapeutic agents that are selected for theirparticular usefulness against the condition that is being treated. Forexample, the compounds of the instant invention may also beco-administered with other well known cancer therapeutic agents that areselected for their particular usefulness against the condition that isbeing treated. Included in such combinations of therapeutic agents arecombinations of the instant farnesyl-protein transferase inhibitors andan antineoplastic agent. It is also understood that such a combinationof antineoplastic agent and inhibitor of farnesyl-protein transferasemay be used in conjunction with other methods of treating cancer and/ortumors, including radiation therapy and surgery.

Examples of an antineoplastic agent include, in general,microtubule-stabilizing agents (such as paclitaxel (also known asTaxol®), docetaxel (also known as Taxotere®), epothilone A, epothiloneB, desoxyepothilone A, desoxyepothilone B or their derivatives);microtubule-disruptor agents; alkylating agents, anti-metabolites;epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes; biologicalresponse modifiers and growth inhibitors; hormonal/anti-hormonaltherapeutic agents and haematopoietic growth factors.

Example classes of antineoplastic agents include, for example, theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the taxanes, the epothilones,discodermolide, the pteridine family of drugs, diynenes and thepodophyllotoxins. Particularly useful members of those classes include,for example, doxorubicin, carminomycin, daunorubicin, aminopterin,methotrexate, methopterin, dichloro-methotrexate, mitomycin C,porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosinearabinoside, podophyllotoxin or podo-phyllotoxin derivatives such asetoposide, etoposide phosphate or teniposide, melphalan, vinblastine,vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like.Other useful antineoplastic agents include estramustine, cisplatin,carboplatin, cyclophosphamide, bleomycin, tamoxifen, ifosamide,melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate,trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11,topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindolederivatives, interferons and interleukins.

The preferred class of antineoplastic agents is the taxanes and thepreferred antineoplastic agent is paclitaxel.

Radiation therapy, including x-rays or gamma rays which are deliveredfrom either an externally applied beam or by implantation of tinyradioactive sources, may also be used in combination with the instantinhibitor of farnesyl-protein transferase alone to treat cancer.

Additionally, compounds of the instant invention may also be useful asradiation sensitizers, as described in WO 97/38697, published on Oct.23, 1997, and herein incorporated by reference.

The instant compounds may also be useful in combination with otherinhibitors of parts of the signaling pathway that links cell surfacegrowth factor receptors to nuclear signals initiating cellularproliferation. Thus, the instant compounds may be utilized incombination with a compound which has Raf antagonist activity. Theinstant compounds may also be co-administered with a compounds that is aselective inhibitor of geranylgeranyl protein transferase type I, aselective inhibitor of farnesyl-protein transferase and/or a compoundthat is a dual inhibitor of geranylgeranyl protein transferase type Iand farnesyl-protein transferase. Such a selective inhibitor or dualinhibitor may be an inhibitor that is competitive with the binding ofthe CAAX-containing protein substrate of farnesyl-protein transferase ormay be a prenyl pyrophosphate compeptitive inhibitor.

In particular, the compounds disclosed in the following patents andpublications may be useful as farnesyl pyrophosphate-competitiveinhibitor component of such a composition: U.S. Ser. Nos. 08/254,228 and08/435,047. Those patents and publications are incorporated herein byreference.

In practicing methods of this invention, which comprise administering,simultaneously or sequentially or in any order, two or more of a proteinsubstrate-competitive inhibitor and a prenyl pyrophosphate-competitiveinhibitor, such administration can be orally or parenterally, includingintravenous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration. It is preferred that suchadministration be orally. It is more preferred that such administrationbe orally and simultaneously. When the protein substrate-competitiveinhibitor and prenyl pyrophosphate-competitive inhibitor areadministered sequentially, the administration of each can be by the samemethod or by different methods.

The instant compounds may also be useful in combination with an integrinantagonist for the treatment of cancer, as described in U.S. Ser. No.09/055,487, filed Apr. 6, 1998, which is incorporated herein byreference.

As used herein the term an integrin antagonist refers to compounds whichselectively antagonize, inhibit or counteract binding of a physiologicalligand to an integrin(s) that is involved in the regulation ofangiogenisis, or in the growth and invasiveness of tumor cells. Inparticular, the term refers to compounds which selectively antagonize,inhibit or counteract binding of a physiological ligand to the αvβ3integrin, which selectively antagonize, inhibit or counteract binding ofa physiological ligand to the αvβ5 integrin, which antagonize, inhibitor counteract binding of a physiological ligand to both the αvβ3integrin and the αvβ5 integrin, or which antagonize, inhibit orcounteract the activity of the particular integrin(s) expressed oncapillary endothelial cells. The term also refers to antagonists of theαvβ6, αvβ8, α1β1, α2β1, α5β, α6β1 and α6β4 integrins. The term alsorefers to antagonists of any combination of αvβ3, αvβ5, αvβ6, αvβ8,α1β1, α2β1, α5β1, α6β1 and α6β4 integrins. The instant compounds mayalso be useful with other agents that inhibit angiogenisis and therebyinhibit the growth and invasiveness of tumor cells, including, but notlimited to angiostatin and endostatin.

Similarly, the instant compounds may be useful in combination withagents that are effective in the treatment and prevention of NF-1,restenosis, polycystic kidney disease, infections of hepatitis delta andrelated viruses and fungal infections.

If formulated as a fixed dose, such combination products employ thecombinations of this invention within the dosage range described belowand the other pharmaceutically active agent(s) within its approveddosage range. Combinations of the instant invention may alternatively beused sequentially with known pharmaceutically acceptable agent(s) when amultiple combination formulation is inappropriate.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof.

Example 1 Preparation Of 1-(4-cyanobenzyl)-5-chloromethyl Imidazole HClSalt

Step 1: Preparation of 4-Cyanobenzylamine

Method 1 (Hydrochloride salt): A 72 liter vessel was charged with 190proof ethanol (14.4 L) followed by the addition of 4-cyanobenzylbromide(2.98 kg) and HMTA (2.18 kg) at ambient temperature. The mixture washeated to about 72-750° C. over about 60 min. On warming, the solutionthickens and additional ethanol (1.0 liter) was added to facilitatestirring. The batch was aged at about 72-75° C. for about 30 min.

The mixture was allowed to cool to about 20° C. over about 60 min, andHCl gas (2.20 kg) was sparged into the slurry over about 4 hours duringwhich time the temperature rose to about 65° C. The mixture was heatedto about 70-72° C. and aged for about 1 hour. The slurry was cooled toabout 30° C. and ethyl acetate (22.3 L) added over about 30 min. Theslurry was cooled to about −5° C. over about 40 min and aged at about −3to about −5° C. for about 30 min. The mixture was filtered and thecrystalline solid was washed with chilled ethyl acetate (3×3 L). Thesolid was dried under an N₂ stream for about 1 hour before charging to a50 liter vessel containing water (5.5 L). The pH was adjusted to about10-10.5 with 50% NaOH (4.0 kg) maintaining the internal temperaturebelow about 30° C. At about 25° C., methylene chloride (2.8 L) was addedand stirring continued for about 15 min. The layers were allowed tosettle and the lower organic layer was removed. The aqueous layer wasextracted with methylene chloride (2×2.2 L). The combined organic layerswere dried over potassium carbonate (650 g). The carbonate was removedvia filtration and the filtrate concentrated in vacuo at about 25° C. togive a free base as a yellow oil.

The oil was transferred to a 50 liter vessel with the aid of ethanol(1.8 L). Ethyl acetate (4.1 L) was added at about 25° C. The solutionwas cooled to about 15° C. and HCl gas (600 g) was sparged in over about3 hours, while keeping batch temperature below about 40° C. At about20-25° C., ethyl acetate (5.8 L) was added to the slurry, followed bycooling to about −5° C. over about 1 hour. The slurry was aged at about−5° C. for about 1 hour and the solids isolated via filtration. The cakewas washed with a chilled mixture of EtOAc/EtOH (9:1 v/v) (1×3.8 L),then the cake was washed with chilled EtOAc (2×3.8 L). The solids weredried in vacuo at about 25° C. to provide the above-titled compound. ¹HNMR (250 MHz, CDCl₃) δ 7.83-7.79 (d, 2H), 7.60-7.57 (d, 2H), 4.79 (s,2H), 4.25 (s, 2H); ¹³C NMR (62.9 MHz, CDCl₃) δ 149.9, 139.8, 134.2,131.2, 119.7, 113.4, 49.9, 49.5, 49.2, 48.8, 48.5, 48.2, 43.8.

Method 2 (phosphate salt): A slurry of HMTA in 2.5 L EtOH was addedgradually over about 30 min to about 60 min to a stirred slurry ofcyanobenzyl-bromide in 3.5 L EtOH and maintained at about 48-53° C. withheating & cooling in a 22L neck flask (small exotherm). Then thetransfer of HMTA to the reaction mixture was completed with the use of1.0 L EtOH. The reaction mixture was heated to about 68-73° C. and agedat about 68-73° C. for about 90 min. The reaction mixture was a slurrycontaining a granular precipitate which quickly settled when stirringstopped.

The mixture was cooled to a temperature of about 50° C. to about 55° C.Propionic acid was added to the mixture and the mixture was heated andmaintained at a temperature of about 50° C. to about 55° C. Phosphoricacid was gradually added over about 5 min to about 10 min, maintainingthe reaction mixture below about 65° C. to form a precipitate-containingmixture. Then the mixture was gradually warmed to about 65° C. to about70° C. over about 30 min and aged at about 65° C. to about 70° C. forabout 30 min. The mixture was then gradually cooled to about 20-25° C.over about 1 hour and aged at about 20-25° C. for about 1 hour.

The reaction slurry was then filtered. The filter cake was washed fourtimes with EtOH, using the following sequence, 2.5 L each time. Thefilter cake was then washed with water five times, using 300 mL eachtime. Finally, the filter cake was washed twice with MeCN (1.0 L eachtime) and the above identified compound was obtained.

Step 2: Preparation of1-(4-Cyanobenzyl)-2-mercapto-5-hydroxymethylimidazole

7% water in acetonitrile (50 mL) was added to a 250 mL roundbottomflask. Next, an amine phosphate salt (12.49 g), as described in Step 1,was added to the flask. Next potassium thiocyanate (6.04 g) anddihydroxyacetone (5.61 g) was added. Lastly, propionic acid (10.0 mL)was added. Acetonitrile/water 93:7 (25 mL) was used to rinse down thesides of the flask. This mixture was then heated to 60° C., aged forabout 30 minutes and seeded with 1% thioimidazole. The mixture was thenaged for about 1.5 to about 2 hours at 60° C. Next, the mixture washeated to 70° C., and aged for 2 hours. The temperature of the mixturewas then cooled to room temperature and was aged overnight. Thethioimidazole product was obtained by vacuum filtration. The filter cakewas washed four times acetonitrile (25 mL each time) until the filtratesbecame nearly colorless. Then the filter cake was washed three timeswith water (approximately 25-50 mL each time) and dried in vacuo toobtain the above-identified compound.

Step 3: Preparation of 1-(4-Cyanobenzyl)-5-Hydroxymethylimidazole

A 1L flask with cooling/heating jacket and glass stirrer (Lab-Max) wascharged with water (200 mL) at 25° C. The thioimidazole (90.27 g), asdescribed in Step 2, was added, followed by acetic acid (120 mL) andwater (50 mL) to form a pale pink slurry. The reaction was warmed to 40°C. over 10 minutes. Hydrogen peroxide (90.0 g) was added slowly over 2hours by automatic pump maintaining a temperature of 35-45° C. Thetemperature was lowered to 25° C. and the solution aged for 1 hour.

The solution was cooled to 20° C. and quenched by slowly adding 20%aqueous Na₂SO₃ (25 mL) maintaining the temperature at less than 25° C.The solution was filtered through a bed of DARCO G-60 (9.0 g) over a bedof SolkaFlok (1.9 g) in a sintered glass funnel. The bed was washed with25 mL of 10% acetic acid in water.

The combined filtrates were cooled to 15° C. and a 25% aqueous ammoniawas added over a 30 minute period, maintaining the temperature below 25°C., to a pH of 9.3. The yellowish slurry was aged overnight at 23° C.(room temperature). The solids were isolated via vacuum filtration. Thecake (100 mL wet volume) was washed with 2×250 mL 5% ammonia (25%) inwater, followed by 100 mL of ethyl acetate. The wet cake was dried withvacuum/N₂ flow and the above-titled compound was obtained.

1H NMR (250 MHz, CDCl3): δ 7.84-7.72 (d, 2H), 7.31-7.28 (d, 2H), 6.85(s, 1H), 5.34 (s, 2H), 5.14-5.11 (t, 1H), 4.30-4.28 (d, 2H), 3.35 (s,1H).

Step 4: Preparation of 1-(4-cyanobenzyl)-5-chloromethyl imidazole HClsalt

Method 1: 1-(4-Cyanobenzyl)-5-hydroxy-methylimidazole (1.0 kg), asdescribed in above in Step 3, was slurried with DMF (4.8 L) at 22 ° C.and then cooled to −5° C. Thionyl chloride (390 mL) was added dropwiseover 60 min during which time the reaction temperature rose to a maximumof 9° C. The solution became nearly homogeneous before the product beganto precipitate from solution. The slurry was warmed to 26° C. and agedfor 1 h.

The slurry was then cooled to 5° C. and 2-propanol (120 mL) was addeddropwise, followed by the addition of ethyl acetate (4.8 L). The slurrywas aged at 5° C. for 1 h before the solids were isolated and washedwith chilled ethyl acetate (3×1 L). The product was dried in vacuo at40° C. overnight to provide the above-titled compound.

¹H NMR (250 MHz DMSO-d₆): δ 9.44 (s, 1H), 7.89 (d, 2H, 8.3 Hz), 7.89 (s,1H), 7.55 (d, 2H, 8.3 Hz), 5.70 (s, 2H), 4.93 (s, 2H). ¹³C NMR (75.5 MHzDMSO-d₆): δ_(c) 139.7, 137.7, 132.7, 130.1, 128.8, 120.7, 118.4, 111.2,48.9, 33.1.

Method 2: To an ice cold solution of dry acetonitrile (3.2 L, 15 L/Kghydroxymethylimidazole) was added 99% oxalyl chloride (101 mL, 1.15 mol,1.15 equiv.), followed by dry DMF (178 mL, 2.30 mol, 2.30 equiv.), atwhich time vigorous evolution of gas was observed. After stirring forabout 5 to 10 min following the addition of DMF, solidhydroxymethylimidazole (213 g, 1.00 mol), as described above in Step 3,was added gradually. After the addition, the internal temperature wasallowed to warm to a temperature of about 23° C. to about 25° C. andstirred for about 1 to 3 hours. The mixture was filtered, then washedwith dry acetonitrile (400 mL displacement wash, 550 mL slurry wash, anda 400 mL displacement wash). The solid was maintained under a N₂atmosphere during the filtration and washing to prevent hydrolysis ofthe chloride by adventitious H₂O. This yielded the crystalline form ofthe chloromethylimidazole hydrochloride.

¹H NMR (250 MHz DMSO-d₆): δ 9.44 (s, 1H), 7.89 (d, 2H, 8.3 Hz), 7.89 (s,1H), 7.55 (d, 2H, 8.3 Hz), 5.70 (s, 2H), 4.93 (s, 2H). ¹³C NMR (75.5 MHzDMSO-d₆): δ_(c) 139.7, 137.7, 132.7, 130.1, 128.8, 120.7, 118.4, 111.2,48.9, 33.1.

Method 3: To an ice cold solution of dry DMF (178 mL, 2.30 mol, 2.30equiv.) in dry acetonitrile (2.56 L, 12 L/Kg Hydroxymethylimidazole) wasadded oxalyl chloride (101 mL, 1.15 mol, 1.15 equiv). The heterogeneousmixture in the reagent vessel was then transferred to a mixture ofhydroxymethylimidazole (213 g, 1.00 mol), as described above in Step 3,in dry acetonitrile (1.7 L, 8 L/Kg hydroxymethylimidazole). Additionaldry acetonitrile (1.1-2.3 L, 5-11 L/Kg hydroxymethylimidazole) was addedto the remaining solid Vilsmeier reagent in the reagent vessel. This,now nearly homogenous, solution was transferred to the reaction vesselat Ti≦+6° C. The reaction vessel temperature was warmed to a temperatureof about 23° C. to about 25° C. and stirred for about 1 to 3 hours. Themixture was then cooled to 0° C. and aged 1 h. The solid was filteredand washed with dry, ice cold acetonitrile (400 mL displacement wash,550 mL slurry wash, and a 400 mL displacement wash). The solid wasmaintained under a N₂ atmosphere during the filtration and washing toprevent hydrolysis of the chloride by adventitious H₂O. This yielded thecrystalline form of the chloromethylimidazole hydrochloride.

Example 2 1-(4′-Cyanobenzyl)imidazol-5-ylmethyl piperazine

Step 1: 1-(4′-Cyanobenzyl)imidazol-5-ylmethyl piperazine-4-carboxylicacid benzyl ester

To an acetonitrile solution of1-(4′-cyanobenzyl)-5-chloromethylimidazole (7.45 mmol) prepared asdescribed in Example 1 and diisopropylethyl amine (22.4 mmol) was added1-benzyl 1-piperazine carboxylate (10.4 mmol). This solution was stirredfor 4.0 hour at 80° C. The product was isolated after silica columnpurification. ¹H—NMR (CDCl-₃): 7.65 (d, 2H); 7.55 (s, 1H); 7.38 (m, 5H);7.15 (d, 2H); 7.0 (s, 1H); 5.3 (s, 2H); 5.1 (s, 1H); 3.4 (m, 4H); 3.3(s, 2H); 2.3 (m, 4H).

Step 2: 1-(4′-Cyanobenzyl)imidazol-5-ylmethyl piperazine

The product from Step 1 (6.17 mmol) was dissolved in absolute ethanolfollowed by the introduction of 10% Pd/C catalyst then hydrogen underatmospheric pressure. The catalyst was removed via filtration throughfilter-aid and the product was isolated by removing the solvent underreduced pressure. ¹H—NMR (CD₃OD): 7.8 (s, 1H); 7.75 (d, 2H); 7.3 (d,2H); 6.9 (s, 1H); 5.45 (s, 2H); 3.3 (m, 4H); 2.6 (s, 2H); 2.3 (m, 4H).

Example 31-(4-Cyanobenzyl)-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

Step 1:4-Benzyloxycarbonyl-[1-(2-oxo-2-(adamant-1-yl))ethyl]piperazin-2-one

To a round bottomed flask were added 4-benzyloxycarbonylpiperazin-2-one(234.26 mg, 1.0 mmol) along with N,N-dimethylforamide(5.0 ml), andsodium hydride (73.0 mg (60%), 1.0 mmol). After the evolution ofhydrogen had ceased the reaction was allowed to stir for an additional30 minutes. To this was added 1 adamantyl bromomethyl ketone(257.18 mg,1.0 mmol). The reaction stirred at Rt. for 18 hrs. The reaction waspoured into water(25ml) and extracted with ethylacetate(2×25 ml). Theethylacetate layer was washed with brine and dried (MgSO₄). Solventremoval yielded4-benzyloxycarbonyl-[1-(2-oxo-2-(adamant-1-yl)ethyl]piperazin-2-one. 400Mhz H¹ NMR (CDCl₃): 1.68-1.91(m,12H), 2.06 (br s,3H), 3.32(t,2H),3.77(t,2H), 4.20(s,2H), 4.34(s,2H), 5.16(s,2H), 7.36(m,5H). The materialwas used with out further purification.

Step 2: 1-[1-(2-oxo-2-(adamant-1-yl))ethyl]piperazin-2-one

4-Benzyloxycarbonyl-[1-(2-oxo-2-(adamant-1-yl))ethyl]piperazin-2-one(389.2 mg, 0.9 mmol) was placed it a parr flask along with palladiumhydroxide on carbon(50 mg, 20 wt. %), Ethanol(75.0 ml), Water(5.0 ml),and 2 drops of conc. HCl. The flask was placed on the Parr apparatus andcharged with 50 psi of hydrogen. The reaction was allowed to shake forfour hours. The reaction was filtered through a celite column and thesolvents removed under high vacuum. This resulted in1-[1-(2-oxo-2-(adamant-1-yl))ethyl]piperazin-2-one. 400 Mhz H¹ NMR(CD₃OD): 1.74-1.83(m,6H), 1.91 (br s,6H), 2.05(br s,3H), 3.58(s,4H),3.90(s,2H), 4.50(s,2H). The material was used with out furtherpurification.

Step 3:1-(4-Cyanobenzyl)-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-[1-(2-Oxo-2-(adamant-1-yl))ethyl]piperazin-2-one (253.4 mg, 0.81 mmol)was placed into a round bottomed flask and diisopropylethylamine (0.71ml), acetonitrile (20.0 ml) and1-(4-cyanobenzyl-5-chloromethyl)imidazole hydrochloride (prepared asdescribed in Example 1) (217.2 mg, 0.81 mmol). The reaction was heatedat reflux for 3 hours. The excess acetonitrile was removed under vacuumand the residue dissolved into ethylacetate(25.0 ml). The ethylacetatelayer was washed with water and the aqueous layer back extracted withadditional ethylacetate(15.0 ml). The combined ethylacetate extractswere dried(MgSO₄). Solvent removal yielded 249.0 mg (65%) of a yellowfoam. The foam was dissolved in to 1.0N HCl and washed withhexane/ethylacetate 25 ml (75/25). The aqueous layer was then basifiedwith ammonium hydroxide and the aqueous layer reextracted withethylacetate. The organic layer was again dried(MgSO₄) and solventremoved. The residue was treated with a dioxane/methanol/HCl solutionwhich gave, after lyophlizing,1-(4-Cyanobenzyl-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazoledihydrochloride. 400 Mhz H¹ NMR (CDCl₃): 1.55-1.75(m,12H), 2.01(brs,3H), 2.40-2.61(m,2H), 3.06-3.19(m,5H), 3.32(s,2H), 3.71-3.75(d,1H),5.29(s,2H), 7.02(s,1H), 7.14(d, 2H), 7.58(s,1H), 7.64(d,2H)

Example 4

R/S1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

A solution of 201 mg (0.4 mmol) of1-(4-cyanobenzyl-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]-imidazole(prepared as described in Example 3), 138.0 mg (1.0 mmol) sodiumcarbonate in methanol 10 ml was treated with sodium borohydride (22.6mg, 0.60 mmol) at 0° C. The reaction was allowed to warm to Rt. Thereaction was diluted with water/Brine and the reaction extracted withethyl acetate. The ethylacetate layer was washed with water/brine anddried (MgSO₄). Solvent removal yielded 189.0 mg of a solid. The solidwas flashed through a small silica column eluting withmethanol/chloroform (10/90). This resulted in R/S1-(4-Cyanobenzyl-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole.400 Mhz H¹ NMR (CDCl₃): 1.55-1.75(m,12H), 2.01(br s,3H),2.40-2.61(m,2H), 3.06-3.19(m,5H), 3.32(s,2H), 3.71-3.75(d,1H),5.29(s,2H), 702(s,1H), 7.14(d,2H), 7.58(s,1H), 7.64(d,2H). High Res. FABMS: Theoretical Mass 474.2864, Measured Mass 474.2864 (C₂₈H₃₅N₅O₂+H⁺)

Example 51-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl]methyllimidazole(Isomer I) and1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole(Isomer II)

R/S1-(4-Cyanobenzyl-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole(prepared as described in Example 3), 50.0 mg(0.1 mmol) was put througha Chiralpack AD column eluting with hexane/ethanol (35/65) Flow Rate 7.0ml/min. The first fraction off was concentrated under high vac to give aglass. The glass was dissolved into dioxane and 2 drops of 1.0N HCl indioxane added. The solution was lyophilized to give a white solid(Isomer I). High Res. FAB MS: Theoretical Mass 474.2863, Measured Mass474.2871 (C₂₈H₃₅N₅O₂+H⁺).

The second fraction off the above column was processed as above to givea white solid (Isomer II). High Res. FAB MS: Theoretical Mass 474.2863,Measured Mass 474.2861 (C₂₈H₃₅N₅O₂+H⁺)

Example 61-(4-Cyanobenzyl)-5-[1-(2-acetyloxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazaole

R/S1-(4-Cyanobenzyl-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole(92.0 mg,0.2 mmol) was dissolved into pyridine (1.0 ml) and the solutioncooled to 5° C. (ice/water). To this solution was added aceticanhydride(20 μl,0.2 mmol). The reaction was allowed to warm to room temperature.The solvent was removed under vac and the residue dissolved into ethylacetate (10 ml), washed with water (1×1.0 ml), brine(1×1.0 ml), anddried (MgSO₄) Solvent removal yielded a tan solid (105.0 mg). The solidwas purified on a prep column (Waters C18). Lyophilizing resulted in awhite solid. 400 Mhz H¹ NMR (CD₃OD): 1.64(br s,6H), 1.74(q,6H), 1.99(brs,3H), 2.02(s,3H), 2.60(m,2H), 2.91(dd,2H), 3.03-3.09(m,1H), 3.27(d,1H),3.39(d,1H), 3.54(dd,2H), 3.65(dd,1H), 4.75(d,1H), 5.65(s,2H),7.44(d,2H), 7.62(s,1H)7.78(d,2H), 9.09(s,1H). High Res. FAB MS:Theoretical Mass 516.2969 , Measured Mass 516.2989(C₃₀H₃₇N₅O₃+H⁺)

Example 71-[l-(4′-Cyanobenzyl)imidazol-5-ylmethyl]piperazine-4-(N-1-adamantyl)carboxamide

To 1-(4′-cyanobenzyl)imidazol-5-yl methyl piperazine Example 2, step 2(0.445 mmol) in ethyl acetate and methanol was added the commerciallyavailable 1-adamantyl isocyanate (0.423 mmol) and dinsopropylethyl amine(0.89 mmol). After stirring for 18 hours the reaction was purified viapreparative hplc and the title compound was then isolated vialyophilization. FAB-MS: calc: 458.6 found: 459.6. ¹H-NMR (CD₃OD): 9.1ppm (7, 1H); 7.85ppm (d, 2H); 7.6 ppm (s, 1H); 7.45 ppm (d, 2H); 5.7 ppm(s, 2H); 3.55 ppm (s, 2H); 3.2ppm (m, 4H); 2.4 ppm (m, 4H); 2.1 ppm (m,9H); 1.7 ppm (s, 6H).

Example 8 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(N-1-adamantyl)carbonyl piperazine

Step 1: 1-(N-boc)-4-(N-1-adamantyl)carbonyl piperazine

Commercially available 1-boc-piperazine (10 mmol) and 1-adamantanecarbonyl chloride (10 mmol) were dissolved in chloroform (50 mL) andDIEA (22 mmol) and stirred for 18 hours. The reaction was then dilutedwith chloroform and washed with dilute potassium hydrogen sulfate,dilute sodium bicarbonate and saturated sodium chloride solutions. Theorganic layer was dried with sodium sulfate and evaporated to providethe title compound. ¹H-NMR (CDCl₃): 3.65 ppm (m, 4H); 3.4 ppm (m, 4H);2.1 ppm (s, 3H); 2.0 ppm (s, 6H); 1.7 ppm (s, 6H); 1.4 ppm (s, 9H).

Step 2:1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(N-1-adamantyl)carbonylpiperazine

The product from Step 1 was treated with neat trifluoroacetic acid andthe desired product was isolated as a free base compound afterpartitioning between methylene chloride and sodium carbonate. Thisproduct (0.373 mmol) was then added to a solution of1-(4′-cyanobenzyl)-5-chloromethylimidazole (1, 0.373 mmol), prepared asdescribed in Example 1, in acetonitrile and DIEA (0.746 mmol) andstirred for 3 hours at 80° C. The product was purified via preperativehplc and the title compound isolated via lyophilization. FAB-MS: calc:443.6 found: 444.3. ¹H-NMR (CD₃OD): 9.1 ppm (s, 1H); 7.8 ppm (d, 2H);7.6 ppm (s, 1H); 7.5 ppm (d, 2H); 5.7 ppm (s, 2H); 3.5 ppm (m, 6H); 2.4ppm (m, 4H); 2.05 ppm (s, 3H); 2.0 ppm (s, 6H); 1.8 ppm (s, 6H).

Example 91-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-[N-(1R)-(−)-10-camphorsulfonyl]piperazine

Step 1:1-(4′-Cyanobenzyl)imidazol-5-ylmethyl-4-[N-(1R)-(−)-10-camphorsulfonyl]piperazine

The commercially available (1R)-(−)-10-camphor-sulfonyl chloride (45,0.359 mmol) and 1-(4′-Cyanobenzyl)imidazol-5-ylmethyl piperazineprepared as described in

Example 2 (0.342 mmol) were dissolved in ethyl acetate and DIEA (0.684mmol) and stirred for 18 hours at 25° C. The product was purified viapreperative hplc and the title compound was isolated via lyophilization.FAB-MS: calc: 495.6 found: 496.3. ¹H-NMR (CD₃OD): 9.1 ppm (s, 1H); 7.9ppm (d, 2H); 7.7 ppm (s, 1H); 7.5 ppm (d, 2H); 5.7 ppm (s, 2H); 3.6 ppm(s, 2H); 3.1 ppm (d, 1H); 3.2 ppm (m, 4H); 2.8 ppm (d, 1H); 2.4 ppm (m,4H); 2.1 ppm (m, 3H); 1.6 ppm (m, 1H); 1.5 ppm (m, 1H); 1.1 ppm (s, 3H);0.9 ppm (s, 3H). Example 101-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(1-adamantylmethyl)piperazine

Step 1: 1-(N-1-adamantyl)methyl piperazine

1-(N-boc)-4-(N-1-adamantyl)carbonyl piperazine (2.98 mmol), prepared asdescribed in

Example 8, Step 1, was treated with neat trifluoroacetic acid and theintermediate was isolated as a free base compound after partitioningbetween methylene chloride and sodium carbonate and then reduced withlithium aluminum hydride (5.96 mmol) in refluxing THF for 18 hours andthen worked up as specified in Fieser and Fieser, 1967, 1, 584, toprovide the titled compound. FAB-MS: calc: 234.6 found: 235.1. ¹H-NMR(CDCl₃): 2.8 ppm (m, 4H); 2.45 ppm (m, 4H); 2.0 ppm (m, 5H); 1.8-1.5 ppm(m, 12H).

Step 2: 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(1-adamantylmethyl)piperazine

The product from Step 1 (0.812 mmol) was then added to a solution of1-(4′-cyanobenzyl)-5-chloromethylimidazole (0.812 mmol), prepared asdescribed in Example 1, in acetonitrile and DIEA (1.62 mmol) and stirredfor 3 hours at 80° C. The product was purified via preperative hplc andthe titled compound was isolated via lyophilization. FAB-MS: calc: 429.5found: 430.2. ¹H-NMR (CD₃OD): 8.3 ppm (s, 1H); 7.8 ppm (d, 2H); 7.4 ppm(d, 2H); 7.3 ppm (s, 1H); 5.6 ppm (s, 2H); 3.5 ppm (s, 2H); 2.5-2.9 ppm(m, 10H); 2.05 ppm (m, 3H); 1.7-1.9 ppm (m, 12H).

Example 111-(4-Cyanobenzyl)-5-[1-(2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

Step 1: 1-Bromo-2-(adamant-1-yl)ethane

1-Hydroxy-2-(adamant-1-yl)ethane (901.0 mg, 5.0 mmol) was treated withphosphorus pentabromide(3.0 gm, 7.0 mmol) in diethyl ether and warmedslightly until a noticeable exotherm was observed. The resulting redsolution was washed with aqueous sodium bicarbonate solution and dried(Na₂SO₄). Solvent removal gave crude 1-Bromo-2-(adamant-1-yl)ethane. Thematerial was used as is without further purification.

Step 2: 4-Benzyloxycarbonyl-[1-(2-(adamant-1-yl))ethyl]piperazin-2-one

4-Benzyloxycarbonylpiperazin-2-one (1.17 g, 5.0 mmol) was dissolved into dry dimethylforamide and sodium hydride (220 mg, 5.5 mmol) added.After the evolution of hydrogen had stopped the1-Bromo-2-(adamant-1-yl)ethane, prepared as described in Step 1, wasadded and the reaction heated at 80° C. for 8 hours. The reaction wasdiluted with water and extracted with methylene chloride. The methylenechloride layer was dried(MgSO₄), and solvent removed to yield a gum. Thegum was dissolved into n-butylchloride and flashed through a shortsilica column. This resulted the title4-Benzyloxycarbonyl-[1-(2-(adamant-1-yl))ethyl]piperazin-2-one. 400 MhzH¹ NMR (CDCl₃): 1.24-1.36(m, 2H), 1.52(s,6H), 1.61-1.77(m,6H), 1.95(brs,3H), 3.10(br s, 2H), 3.69(t,2H), 4.12(s,2H), 5.15(d,2H),7.35-7.38(m,5H). High res. ES MS: Theoretical Mass 397.2486, MeasuredMass 397.2502 (C₂₄H₃₂N₂O₃+H⁺)

Step 3: 1-[(2-(adamant-1-yl))ethyl]piperazin-2-one

4-Benzyloxycarbonyl-[1-(2-(adamant-1-yl))ethyl]piperazin-2-one (570.0mg, 1.44 mmol), prepared as described in Step 2, was added to a flaskcontaining ethanol(15.0 ml), water(1.0 ml), conc. hydrochloric acid(2drops, and palladium hydroxide(20% by Wt., 50.0 mg). The flask washydrogenated for 3.5 hours. The reaction was filtered through celite andthe solvents removed to give 1-[(2-(adamant-1-yl))ethyl]piperazin-2-oneas the hydrochloride salt. 400 Mhz H¹ NMR (CD₃OD): 1.33-1.37(m, 2H),1.58(s,6H), 1.73(q, 6H), 3.45-3.53(m,4H), 3.61(t, 2H), 3.80(s,2H). Highres. FAB MS: Theoretical Mass 263.2118, Measured Mass5263.2122(C₁₆H₂₆N₂O+H⁺)

Step 4:1-(4-Cyanobenzyl)-5-[1-(2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-[(2-(adamant-1-yl))ethyl]piperazin-2-one hydrochloride (400.0 mg, 1.34mmol), prepared as described in Step 3, along with1-(4-cyanobenzyl-5-chloromethyl)imidazole hydrochloride (360.0 mg, 1.34mmol), prepared as described in Example 1, acetonitrile(20.0 ml), anddiisopropylethylamine (1.40 ml, 8.0 mmol) were heated at reflux foreight hours. The acetonitrile was removed under vacuum and the residuedissolved in ethyl acetate(50.0 ml). The ethyl acetate solution waswashed with water (2×,10.0 ml), brine(25.0 ml), and dried(MgSO₄). Thesolvents were removed under high vac. The resulting gum purified using asilica column eluting with methanol/chloroform (10/90). The isolatedfree base was dissolved into HCl/dioxane (0.1N, 2.0 ml) and lyophilizedto give1-(4-Cyanobenzyl-5-[1-(2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazoleas the dihydrochloride salt. 400 Mhz H¹ NMR (CDCl₃) (Free Base):1.22-1.26(m, 2H), 1.53(s,6H), 1.67(q, 6H), 1.95(br s,3H), 2.52(t, 2H),3.03(s,2H), 3.05(t,2H), 3.30(s, 2H), 3.31-3.35(m, 2H), 5.29(s, 2H),7.02(s, 1H), 7.13(d, 2H), 7.57(s, 1H), 7.63(d,2H). High res. FAB MS:Theoretical Mass 458.2914, Measured Mass 458.2911(C₂₈H₃₅N₅O+H⁺)

Example 121-(4-Cyanobenzyl)-5-[1-(1-(adamant-1-yl)methyl)-2-oxo-piperazin-4-yl-methyl]imidazole

Step 1:N¹-tert-butylcarboxy-[N²-(adamant-1-yl)methyl)]-1,2-ethanediamine

Boc-aminoacetaldehyde (1.0 g, 6.10 mmol) along with1-adamantanemethylamine(1.0 g, 6.10), and methanol(50.0 ml) werecombined in a round bottom flask. The reaction was cooled to 5° C. in anice/water bath, purged with argon and sodium acetate(1.0 g, 12.20 mmol)added portion wise. Palladium on activated carbon(50.0 mg, Palladiumcontent 10%) was added, the flask charged with hydrogen, and the icebath removed. The reaction was allowed to run over night. The reactionwas purged with argon and filtered through celite. The solvents wereremoved and the resulting gum dissolved in ethyl acetate(100.0 ml). Theethyl acetate solution was extracted with dilute citric acid(2×50.0 ml).The combined citric acid extract was washed with additional ethylacetate(2×25ml). The citric acid solution was then basified using conc.ammonium hydroxide and extracted with chloroform(2×50ml). The chloroformextract was washed with brine(25.0 ml) and dried(MgSO₄). Solvent removalunder high vac resulted inN¹-tert-butylcarboxy-[N²-(adamant-1-yl)methyl)]-1,2-ethanediamine. 400Mhz H¹ NMR (CDCl₃) (Free Base): 1.45(s, 9H), 1.51(s,6H), 1.68(q, 6H),1.96(br s,3H), 2.22(s, 2H), 2.70(t, 2H), 3.20(m,2H). High res. FAB MS:Theoretical Mass 309.2536, Measured Mass 309.2535(C₁₈H₃₂N₂O₂+H⁺)

Step 2:N¹-tert-butylcarboxy[N²-(adamant-1-yl)methyl)]—N²(2-bromoacetyl)-1,2-ethanediamine

N¹-tert-butylcarboxy-[N²-(adamant-1-yl)methyl)]-1,2-ethanediamine (1.20g, 3.90 mmol), prepared as described in Step 1, was placed in a roundbottom flask and ethyl acetate added(50.0 ml), saturated sodiumcarbonate solution(25.0 ml), and water(25.0 ml). The reaction was cooledto 5° C. (ice/water) and bromoacetylbromide added in small portionsuntil no starting material remained(TLC, 95/5, Chloroform/methanol, I₂stain).

The reaction was allowed to warm to room temperature and transferred toa separatory funnel where the mixture was shaken several separate timesuntil the ph of the ethyl acetate layer remained basic. Additional ethylacetate was added(50.0 ml) and the layers were separated. The organiclayer washed with dilute citric acid solution(1×25.0 ml), saturatedsodium bicarbonate(1×25.0 ml), water(1×25.0 ml), brine(1×25.0 ml), anddried(MgSO₄). Solvent removal gaveN¹-tert-butylcarboxy[N²-(adamant-1-yl)methyl)]-N²(2-bromoacetyl)-1,2-ethanediamine.400 Mhz H¹ NMR (CDCl₃): 1.45(s, 9H), 1.5(d,5H), 1.61-1.74(, 7H),1.91(d,3H), 3.08(s, 2H), 3.23-3.39(m, 2H), 3.50-3.54(m,2H), 3.93(s,2H).High res. FAB MS: Theoretical Mass 429.1747, Measured Mass429.1737(C₂₀H₃₃BrN₂O₃+H⁺)

Step 3: 4-Benzyloxycarbonyl-[1-((adamant-1-yl)methyl)]piperazin-2-one

N¹-tert-Butylcarboxy[N²-(adamant-1-yl)methyl)]-N²-(2-bromoacetyl)-1,2-ethanediamine(1.41g,3.3 mmol), prepared as described in Step 2, was added to a reactionflask containing dimethylforamide (25.0 ml), and Cesium carbonate(6.42g, 19.7 mmol). The reaction was heated at 100° C. for ten hours. Thereaction was diluted water/brine (50:50, 50.0 ml) and extracted withethyl acetate(2×50.0 ml). The ethyl acetate layer was then washed withdilute citric acid solution(25.0 ml), saturated sodium bicarbonatesolution(25.0 ml), brine(25.0 ml), and dried(MgSO₄). Solvent removalgave 4-Benzyloxycarbonyl-[1-((adamant-1-yl)methyl)]piperazin-2-one.400Mhz H¹ NMR (CDCl₃): 1.48(s, 9H), 1.52(s,6H), 1.67(q, 6H), 1.97(br s,3H),3.10(s, 2H), 3.39-3.43(m, 2H), 3.60(t,2H), 4.07(s,2H). High res. FAB MS:Theoretical Mass 349.2486, Measured Mass 349.2479(C₂₀H₃₂N₂O₃+H⁺).

Step 4: [1-((adamant-1-yl)methyl)]piperazin-2-one

4-Benzyloxycarbonyl-[1-((adamant-1-yl)methyl)]-piperazin-2-one (300.0mg, 0.9 mmol), prepared as described in Step 3, was dissolved into neattrifluoroacetic acid(5.0 ml) and the reaction allowed to stir for 30minutes. Excess trifluoroacetic acid was removed under high vacuum andthe resulting gum treated with 4.0N HCl in dioxane and after 10 minutesexcess solvents were removed under high vacuum to give1-((adamant-1-yl)methyl)piperazin-2-one as the hydrochloride salt. 400Mhz H¹ NMR (CD3OD): 1.62(s, 6H),1.68-1.77 (m,6H), 1.97(br s,3H), 3.16(s,2H), 3.52(t, 2H), 3.72(t,2H), 3.86(s, 2H). High res. FAB MS: TheoreticalMass 249.1961, Measured Mass 249.1952(C₁₅H₂₄N₂O+H⁺).

Step 5:1-(4-Cyanobenzyl)-5-[1-(1-(adamant-1-yl)methyl)-2-oxo-piperazin-4-yl-methyl]imidazole

1-(4-cyanobenzyl-5-chloromethyl)imidazole hydrochloride(268.0 mg, 1.0mmol), prepared as described in Example 1, was added to a reaction flaskalong with aceto-nitrile(25.0 ml), diisopropylethylamine(1.0 ml, 6.0mmol) and 1-((adamant-1-yl)methyl)piperazin-2-one(275.0 mg, 1.0 mmol)prepared as described in Step 4. The reaction was heated at 80° C. for10 hours. The acetonitrile was removed under high vacuum to give 542.0mg of a foam. The foam was dissolved into ethyl acetate(50.0 ml) andwashed with dilute ammonium hydroxide solution(2×25.0 ml). The organiclayer was washed with brine(25.0 ml), and dried(MgSO₄). Solvent removalgave 382.0 mg of a foam which was flashed through a silica columneluting with methanolchloroform (5/95) to give1-(4-Cyanobenzyl-5-[1-(1-(adamant-1-yl)methyl)-2-oxo-piperazin-4-yl-methyl]imidazoleas the free base. The free base was dissolved into 4.0N dioxane and thesolvents removed under high vacuum to give the title compound as thedihydrochloride salt. 400 Mhz H¹ NMR (CDCl₃) (Free Base): 1.52(s, 6H),1.65(q, 6H), 1.96(s, 3H), 2.53(t, 2H), 3.00(s, 2H), 3.09(s, 2H), 3.18(t,2H), 3.32(s, 2H), 5.31(s, 2H), 7.01(s, 1H), 7.14(d, 2H), 7.56(s, 1H),7.63(d, 2H). High res. FAB MS: Theoretical Mass 444.2758, Measured Mass444.2757(C₂₇H₃₃N₅O+H⁺)

Example 13 1-(4′-Cyanobenzyl)-2-methyl-imidazol-5-ylmethylpiperazine-4-(N-1-adamantyl)carboxamide

Step 1: Preparation of1-(4-Cyanobenzyl)-2-methylimidazole-5-carboxaldehyde

Step A: Preparation of 4-Bromo-2-methylimidazole-5-carboxaldehyde

4-Bromo-5-hydroxymethyl-2-methylimidazole was prepared according to theprocedure described by S. P. Watson, Synthetic Communications, 22,2971-2977 (1992). A solution of4-bromo-5-hydroxymethyl-2-methylimidazole (4.18 g, 21.9 mmol) wasrefluxed with manganese dioxide (16.1 g) in 1:1 methylenechloride:dioxane (200 mL) for 16 h. The cooled reaction was filteredthrough celite and concentrated to yield the title compound as a paleyellow solid.

¹H NMR (CDCl₃, 300 MHz) 8 9.57 (1H, s), 2.52 (3H, s).

Step B: 4-Bromo-1-(4-cyanobenzyl)-2-methylimidazole-5-carboxaldehyde

4-Cyanobenzylbromide (1.05 g, 5.39 mmol) was added to a solution of4-bromo-2-methylimidazole-5-carboxaldehyde (1.02 g, 5.39 mmol) (Step A)in dimethylacetamide (15 mL). The solution was cooled to −10° C. andpowdered potassium carbonate (0.745 g, 5.39 mmol) added. The reactionwas stirred at −10° C. for 2 h, and a further 4 h at 20° C. The reactionwas diluted with water and extracted with ethyl acetate. The organicphase was washed with water, saturated brine, and dried over magnesiumsulfate. Solvent evaporation yielded a white solid. ¹H NMR(CDCl_(3, 400) MHz) 8 9.68 (1H, s), 7.64 (2H, d, J=7 Hz), 7.15 (2H, d,J=7Hz), 5.59 (2H, s), 2.40 (3H, s).

Step C: 1-(4-Cyanobenzyl)−2-methylimidazole-5-carboxaldehyde

A solution of4-bromo-1-(4-cyanobenzyl)-2-methylimidazole-5-carboxaldehyde (1.33 g,4.37 mmol) (Step B) and imidazole (0.600 g, 8.74 mmol) in 1:1 ethylacetate-alcohol (150 mL) was stirred with 10% palladium on carbon (0.020g) under 1 atm hydrogen. After 2 h, the reaction was filtered throughcelite and concentrated to give the title compound as a white solid.

¹H NMR (DMSO-d₆, 400 MHz): 8 9.62 (1 H, s), 7.90 (1H, s), 7.81 (2H, d,J=8 Hz), 7.20 (2H, d, J=8 Hz), 5.64 (2H, s), 2.33 (3H, s).

Step 2: 1-[1-(4-cyanobenzyl)-2-methylimidazol-5-ylmethyl]-4-piperazine

A mixture of 2.50 g (11.05 mmol) of1-(4-cyanobenzyl)-2-methylimidazole-5-carboxaldehyde (prepared asdescribed in Step 1), 2.44 g (11.05 mmol) of1-(benzyloxycarbonyl)piperazine, and 4.11 ml (13.81 mmol) of titaniumisopropoxide in 5 ml anh. THF was stirred at RT for 1 h. The reactionwas diluted with 5 ml of anh. EtOH, and the solution treated with 695mg(11.05 mmol) of sodium cyanoborohydride. The reaction was heated at65° C. for 1 h, and was stirred at RT for an additional 18h. Thereaction was poured into excess water, and the suspension stirredvigorously for 10 min. The mixture was diluted with ethyl acetate, andwas filtered through a Celite pad. The filtrate was separated, and theorganic layer dried and concentrated in vacuo to give a brown oil. Theoil was purified by column chromatography over silica gel usingacetonitrile followed by 5% methanol/chloroform to give 2 g of a yellowoil. The oil was dissolved in 15 ml abs. Ethanol, and was hydrogenatedat atmospheric pressure over 250 mg of 10% Pd on C catalyst. Afterapprox. 30 h, the reaction was filtered through a celite pad to give thedesired product as a pale yellow oil. H1 NMR(CDCl₃): 2.29(s,3H), 2.30(brs, 4H), 2.90(br s, 4H), 3.29(s,2H), 5.14(s,2H), 6.87(s,1H), 7.05(d,2H),7.62(d,2H).

Step 3: 1-(4′-Cyanobenzyl)-2-methyl-imidazol-5-ylmethylpiperazine-4-(N-1-adamantyl)carboxamide (68)

To 1-(4′-Cyanobenzyl)-2-methyl-imidazol-5-ylmethyl piperazine (preparedas described in Step 2) (0.254 mmol) in methylene chloride was added thecommercially available 1-adamantyl isocyanate (0.254 mmol) anddiisopropylethyl amine (0.508 mmol). After stirring for 1 hour thereaction was evaporated and purified via preparative hplc and isolatedvia lyophilization. HI-RES MS: calc: 473.3023 found: 473.3034. 1H-NMR(CD₃OD): 7.75 ppm (d, 2H); 7.3 ppm (m, 3H); 5.55 ppm (s, 2H); 3.35 ppm(s, 2H); 3.1 ppm (m, 4H); 2.5 ppm (s, 3H); 2.35 ppm (m, 4H); 2.05 ppm(s, 3H); 2.0 ppm (s, 6H), 1.65 ppm (s, 6H).

Example 14 Preparation of Carbamates from Alcohols

To each of 14 previously tared screw cap test tubes was addedapproximately 1.25 equivalents of one of the 14 alcohols. Afterdetermining the weights of alcohols by difference the appropriate amountof p-nitrophenyl chloroformate (1.27 equivalents) was added to each ofthe tubes as a freshly made tetrahydrofuran:acetonitrile (7:1) solution.This was stirred for 10 minutes and then pyridine (1.37 equivalents) wasadded to each tube followed by stirring for 1.5 hours. Each tube wasdiluted with 1.5 mL each of ethyl acetate and water. The aqueous wasremoved and the organic layer was rotary speed evaporated to a pellet.To each of the 14 tubes was added 1.0 mL of ethyl acetate:DCM:methanol(1:1:0.1) and 0.1 mmol of a stock DMF solution of1-(4′-Cyanobenzyl)imidazol-5-ylmethyl piperazine, prepared as describedin Example 2 and 0.2 mmol DIEA. Stir for 18 hours. To each tube wasadded 1.0 mL of ethyl acetate then each tube was washed with 5×1 mL of1.0N NaOH. The organic layer was then rotary speed evaporated to apellet. The resulting pellet was dissolved in 2.0 mL of DMSO andsubmitted for LC and HI-RES Mass spec analysis. The completeness ofreaction was estimated by comparing the AUC of each reaction against anindependently prepared standard of known concentration that was alsoprepared in the library run.

The following compounds of the invention were prepared by this method:

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl] piperazine-4-carboxylic acid(2-norbornane)methyl ester

Hi-Res MS: calc: 434.2530 found: 434.2550

[1-(1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-piperazine-4-carboxylic acid(2-norbornane)methyl ester Hi-Res MS: calc: 420.2390 found: 420.2394

Example 15 Preparation of Amides from Acids

To each of twelve previously tared screw cap test tubes was addedapproximately 0.12 mmol of a multicyclic containing carboxylic acidacids. After determining this weight by difference, a DCM solution of2,3-dimethyl-2-fluoro-pyridinium tosylate (1.2 equivalents vs. acid, 218mg/mL) was added to each tube and then a DCM solution of triethyl amine(1.2 equivalents vs. acid, 250 uL/mL DCM) was added. These solutionswere stirred for 15 minutes and then a solution of 1-(4′-cyanobenzyl)imidazol-5-ylmethyl piperazine (0.1 mmol in DMF), prepared as describedin Example 2, and DIEA (0.2 mmol) in DCM (0.5 mL) for a total volume of0.615 mL was added to each tube and then stirred for 5 hours. Each tubewas washed with 2×1 mL of 1% trifluoroacetic acid. The DCM layers wereremoved and then transferred to new pre-tared test tubes and then rotaryspeed evaporated to a pellet. The resulting pellet was dissolved in 2.0mL of DMSO and submitted for LC and HI-RES Mass spec analysis. Thecompleteness of reaction was estimated by comparing the AUC of eachreaction against an independently prepared standard of knownconcentration that was also prepared in the library run.

The following compounds of the invention were prepared by this method:

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]piperazine-4-(2-bicyclo-[2.2.2]-octylcarbonyl)

Hi-Res MS: calc: 418.2613 found: 418.2601

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-piperazine-4-(2-norbornanecarbonyl)

Hi-Res MS: calc: 404.2461 found: 404.2445

1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-cis/trans-(2,6,6-trimethylbicyclo[3.1.1]heptanecarbonyl)-piperazine

Hi-Res MS: calc: 446.291 found: 446.2914

Example 16 In Vitro inhibition of Ras Farnesyl Transferase

Transferase Assays. Isoprenyl-protein transferase activity assays arecarried out at 30° C. unless noted otherwise. A typical reactioncontains (in a final volume of 50 μL): [³H]farnesyl diphosphate, Rasprotein, 50 mM HEPES, pH 7.5, 5 mM MgCl₂, 5 mM dithiothreitol, 10 μMZnCl₂, 0.1% polyethyleneglycol (PEG) (15,000-20,000 mw) andisoprenyl-protein transferase. The FPTase employed in the assay isprepared by recombinant expression as described in Omer, C. A., Kral, A.M., Diehl, R. E., Prendergast, G. C., Powers, S., Allen, C. M., Gibbs,J. B. and Kohl, N. E. (1993) Biochemistry 32:5167-5176. After thermallypre-equilibrating the assay mixture in the absence of enzyme, reactionsare initiated by the addition of isoprenyl-protein transferase andstopped at timed intervals (typically 15 min) by the addition of 1 M HClin ethanol (1 mL). The quenched reactions are allowed to stand for 15 m(to complete the precipitation process). After adding 2 mL of 100%ethanol, the reactions are vacuum-filtered through Whatman GF/C filters.Filters are washed four times with 2 mL aliquots of 100% ethanol, mixedwith scintillation fluid (10 mL) and then counted in a Beckman LS3801scintillation counter.

For inhibition studies, assays are run as described above, exceptinhibitors are prepared as concentrated solutions in 100% dimethylsulfoxide and then diluted 20-fold into the enzyme assay mixture.Substrate concentrations for inhibitor IC₅₀ determinations are asfollows: FTase, 650 nM Ras—CVLS (SEQ.ID.NO.: 1), 100 nM farnesyldiphosphate.

The compounds of the instant invention described in the above Examples3-15 were tested for inhibitory activity against human FPTase by theassay described above and were found to have IC₅₀ of <5 μM.

Example 17 ModifiedIn Vitro GGTase Inhibition Assay

The modified geranylgeranyl-protein transferase inhibition assay iscarried out at room temperature. A typical reaction contains (in a finalvolume of 50 μL): [³H]geranylgeranyl diphosphate, biotinylated Raspeptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10 mMglycerophosphate or 5 mM ATP), 5 mM MgCl₂, 10 μM ZnCl₂, 0.1% PEG(15,000-20,000 mw), 2 mM dithiothreitol, and geranylgeranyl-proteintransferase type I(GGTase). The GGTase-type I enzyme employed in theassay is prepared as described in U.S. Pat. No. 5,470,832, incorporatedby reference. The Ras peptide is derived from the K4B-Ras protein andhas the following sequence: biotinyl-GKKKKKKSKTKCVIM (single amino acidcode) (SEQ.ID.NO.: 2). Reactions are initiated by the addition of GGTaseand stopped at timed intervals (typically 15 min) by the addition of 200μL of a 3 mg/mL suspension of streptavidin SPA beads (ScintillationProximity Assay beads, Amersham) in 0.2 M sodium phosphate, pH 4,containing 50 mM EDTA, and 0.5% BSA. The quenched reactions are allowedto stand for 2 hours before analysis on a Packard TopCount scintillationcounter.

For inhibition studies, assays are run as described above, exceptinhibitors are prepared as concentrated solutions in 100% dimethylsulfoxide and then diluted 25-fold into the enzyme assay mixture. IC₅₀values are determined with Ras peptide near KM concentrations. Enzymeand substrate concentrations for inhibitor IC₅₀ determinations are asfollows: 75 pM GGTase-I, 1.6 μM Ras peptide, 100 nM geranylgeranyldiphosphate.

The compounds of the instant invention described in the above Examples3-15 were tested for inhibitory activity against human GTase type I bythe assay described above and were found to have IC₅₀ of<5 μM.

Example 18 Cell-basedin Vitro Ras Farnesylation Assay

The cell line used in this assay is a v-ras line derived from eitherRatl or NIH3T3 cells, which expressed viral Ha-ras p21. The assay isperformed essentially as described in DeClue, J. E. et al., CancerResearch 51:712-717, (1991). Cells in 10 cm dishes at 50-75% confluencyare treated with the test compound (final concentration of solvent,methanol or dimethyl sulfoxide, is 0.1%). After 4 hours at 37° C., thecells are labeled in 3 ml methionine-free DMEM supple-mented with 10%regular DMEM, 2% fetal bovine serum and 400 μCi[³⁵S]methionine (1000Ci/mmol). After an additional 20 hours, the cells are lysed in 1 mllysis buffer (1% NP40/20 mM HEPES, pH 7.5/5 mM MgCl₂/1 mM DTT/10 mg/mlaprotinen/2 mg/ml leupeptin/2 mg/ml antipain/0.5 mM PMSF) and thelysates cleared by centrifugation at 100,000×g for 45 min. Aliquots oflysates containing equal numbers of acid-precipitable counts are boughtto 1 ml with IP buffer (lysis buffer lacking DTT) andimmuno-precipitated with the ras-specific monoclonal antibody Y13-259(Furth, M. E. et al., J. Virol. 43:294-304, (1982)). Following a 2 hourantibody incubation at 4° C., 200 μl of a 25% suspension of proteinA-Sepharose coated with rabbit anti rat IgG is added for 45 min. Theimmuno-precipitates are washed four times with IP buffer (20 nM HEPES,pH 7.5/1 mM EDTA/1% Triton X-100.0.5% deoxycholate/0.1%/SDS/0.1 M NaCl)boiled in SDS-PAGE sample buffer and loaded on 13% acrylamide gels. Whenthe dye front reached the bottom, the gel is fixed, soaked inEnlightening, dried and autoradiographed. The intensities of the bandscorresponding to farnesylated and nonfarnesylated ras proteins arecompared to determine he percent inhibition of farnesyl transfer toprotein.

Example 19 Cell-basedin Vitro Growth Inhibition Assay

To determine the biological consequences of FPTase inhibition, theeffect of the compounds of the instant invention on theanchorage-independent growth of Ratl cells transformed with either av-ras, v-raf, or v-mos oncogene is tested. Cells transformed by v-Rafand v-Mos maybe included in the analysis to evaluate the specificity ofinstant compounds for Ras-induced cell transformation.

Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded ata density of 1×10⁴ cells per plate (35 mm in diameter) in a 0.3% topagarose layer in medium A (Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum) over a bottom agarose layer(0.6%). Both layers contain 0.1% methanol or an appropriateconcentration of the instant compound (dissolved in methanol at 1000times the final concentration used in the assay). The cells are fedtwice weekly with 0.5 ml of medium A containing 0.1% methanol or theconcentration of the instant compound. Photomicrographs are taken 16days after the cultures are seeded and comparisons are made.

Example 20 Construction of SEAP Reporter Plasmid pDSE100

The SEAP reporter plasmid, pDSE100 was constructed by ligating arestriction fragment containing the SEAP coding sequence into theplasmid pCMV-RE-AKI. The SEAP gene is derived from the plasmidpSEAP2-Basic (Clontech, Palo Alto, Calif.). The plasmid pCMV-RE-AKI wasconstructed by Deborah Jones (Merck) and contains 5 sequential copies ofhe ‘dyad symmetry response element’ cloned upstream of a ‘CAT-TATA’sequence derived from the cytomegalovirus immediate early promoter. Theplasmid also contains a bovine growth hormone poly-A sequence.

The plasmid, pDSE100 was constructed as follows. A restriction fragmentencoding the SEAP coding sequence was cut out of the plasmidpSEAP2-Basic using the restriction enzymes EcoR1 and HpaI. The ends ofthe linear DNA fragments were filled in with the Klenow fragment of E.coli DNA Polymerase I. The ‘blunt ended’ DNA containing the SEAP genewas isolated by electrophoresing the digest in an agarose gel andcutting out the 1694 base pair fragment. The vector plasmid pCMV-RE-AKIwas linearized with the restriction enzyme Bgl-II and the ends filled inwith Klenow DNA Polymerase I. The SEAP DNA fragment was blunt endligated into the pCMV-RE-AKI vector and the ligation products weretransformed into DH5-alpha E. coli cells (Gibco-BRL). Transformants werescreened for the proper insert and then mapped for restriction fragmentorientation. Properly oriented recombinant constructs were sequencedacross the cloning junctions to verify the correct sequence. Theresulting plasmid contains the SEAP coding sequence downstream of theDSE and CAT-TATA promoter elements and upstream of the BGH poly-Asequence.

Alternative Construction of SEAP reporter plasmid. pDSE101

The SEAP reporter plasmid, pDSE101 is also constructed by ligating arestriction fragment containing the SEAP coding sequence into theplasmid pCMV-RE-AKI. The SEAP gene is derived from plasmidpGEM7zf(−)/SEAP.

The plasmid pDSE101 was constructed as follows: A restriction fragmentcontaining part of the SEAP gene coding sequence was cut out of theplasmid pGEM7zf(−)/SEAP using the restriction enzymes Apa I and KpnI.The ends of the linear DNA fragments were chewed back with the Klenowfragment of E. coli DNA Polymerase I. The “blunt ended” DNA containingthe. truncated SEAP gene was isolated by electrophoresing the digest inan agarose gel and cutting out the 1910 base pair fragment. This 1910base pair fragment was ligated into the plasmid pCMV-RE-AKI which hadbeen cut with Bgl-II and filled in with E. coli Klenow fragment DNApolymerase. Recombinant plasmids were screened for insert orientationand sequenced through the ligated junctions. The plasmid pCMV-RE-AKI isderived from plasmid pCMVIE-AKI-DHFR (Whang, Y., Silberklang, M.,Morgan, A., Munshi, S., Lenny, A. B., Ellis, R. W., and Kieff, E. (1987)J. Virol., 61, 1796-1807) by removing an EcoRI fragment containing theDHFR and Neomycin markers. Five copies of the fos promoter serumresponse element were inserted as described previously (Jones, R. E.,Defeo-Jones, D., McAvoy, E. M., Vuocolo, G. A., Wegrzyn, R. J., Haskell,K. M. and Oliff, A. (1991) Oncogene, 6, 745-751) to create plasmidpCMV-RE-AKI.

The plasmid pGEM7zf(-)/SEAP was constructed as follows. The SEAP genewas PCRed, in two segments from a human placenta cDNA library (Clontech)using the following oligos.

Sense strand N-terminal SEAP: 5′ GAGAGGGAATTCGGGCCCTTCCTGCATGCTGCTGCTGCTGCTGCTGCTGGGC 3′ (SEQ.ID.NO.:3)

Antisense strand N-terminal SEAP: 5′ GAGAGAGCTCGAGGTTAACCCGGGTGCGCGGCGTCGGTGGT 3′ (SEQ.ID.NO.:4)

Sense strand C-terminal SEAP: 5′ GAGAGAGTCTAGAGTTAACCCGTGGTCCCCGCGTTGCTTCCT 3′ (SEQ.ID.NO.:5)

Antisense strand C-terminal SEAP: 5′ GAAGAGGAAGCTTGGTACCGCCACTGGGCTGTAGGTGGTGGCT 3′ (SEQ.ID.NO.:6)

The N-terminal oligos (SEQ.ID.NO.: 4 and SEQ.ID.NO.: 5) were used togenerate a 1560 bp N-terminal PCR product that contained EcoRI and HpaIrestriction sites at the ends. The Antisense N-terminal oligo(SEQ.ID.NO.: 4) introduces an internal translation STOP codon within theSEAP gene along with the HpaI site. The C-terminal oligos (SEQ.ID.NO.: 5and SEQ.ID.NO.: 6) were used to amplify a 412 bp C-terminal PCR productcontaining HpaI and HindIII restriction sites. The sense strandC-terminal oligo (SEQ.ID.NO.: 5) introduces the internal STOP codon aswell as the HpaI site. Next, the N-terminal amplicon was digested withEcoRI and HpaI while the C-terminal amplicon was digested with HpaI andHindIII. The two fragments comprising each end of the SEAP gene wereisolated by electrophoresing the digest in an agarose gel and isolatingthe 1560 and 412 base pair fragments. These two fragments were thenco-ligated into the vector pGEM7zf(−) (Promega) which had beenrestriction digested with EcoRI and HindIII and isolated on an agarosegel. The resulting clone, pGEM7zf(−)/SEAP contains the coding sequencefor the SEAP gene from amino acids.

Construction of a constitutively expressing SEAP plasmid pCMV-SEAP

An expression plasmid constitutively expressing the SEAP protein wascreated by placing the sequence encoding a truncated SEAP genedownstream of the cytomegalovirus (CMV) IE-1 promoter. The expressionplasmid also includes the CMV intron A region 5′ to the SEAP gene aswell as the 3′ untranslated region of the bovine growth hormone gene 3′to the SEAP gene.

The plasmid pCMVIE-AKI-DHFR (Whang et al, 1987) containing the CMVimmediate early promoter was cut with EcoRI generating two fragments.The vector fragment was isolated by agarose electrophoresis andreligated. The resulting plasmid is named pCMV-AKI. Next, thecytomegalovirus intron A nucleotide sequence was inserted downstream ofthe CMV IE1 promter in pCMV-AKI. The intron A sequence was isolated froma genomic clone bank and subcloned into pBR322 to generate plasmidp16T-286. The intron A sequence was mutated at nucleotide 1856(nucleotide numbering as in Chapman, B. S., Thayer, R. M., Vincent, K.A. and Haigwood, N. L., Nuc.Acids Res. 19, 3979-3986) to remove a Saclrestriction site using site directed mutagenesis. he mutated intron Asequence was PCRed from the plasmid p16T-287 sing the following oligos.

Sense strand: 5′ GGCAGAGCTCGTTTAGTGAACCGTCAG 3′ (SEQ.ID.NO.: 7)

Antisense strand: 5′ GAGAGATCTCAAGGACGGTGACTGCAG 3′ (SEQ.ID.NO.: 8)

These two oligos generate a 991 base pair fragment with a SacI siteincorporated by the sense oligo and a Bgl-II fragment incorporated bythe antisense oligo. The PCR fragment is trimmed with Sacd and Bgl-IIand isolated on an agarose gel. The vector pCMV-AKI is cut with Sacl andBgl-II and the larger vector fragment isolated by agarose gelelectrophoresis. The two gel isolated fragments are ligated at theirrespective SacI and Bgl-II sites to create plasmid pCMV-AKI-InA.

The DNA sequence encoding the truncated SEAP gene is inserted into thepCMV-AKI-InA plasmid at the Bgl-II site of the vector.

The SEAP gene is cut out of plasmid pGEM7zf(−)/SEAP (described above)using EcoRI and HindIII. The fragment is filled in with Klenow DNApolymerase and the 1970 base pair fragment isolated from the vectorfragment by agarose gel electrophoresis. The pCMV-AKI-InA vector isprepared by digesting with Bgl-II and filling in the ends with KlenowDNA polymerase. The final construct is generated by blunt end ligatingthe SEAP fragment into the pCMV-AKI-InA vector. Transformants werescreened for the proper insert and then mapped for restriction fragmentorientation. Properly oriented recombinant constructs were sequencedacross the cloning junctions to verify the correct sequence. Theresulting plasmid, named pCMV-SEAP, contains a modified SEAP sequencedownstream of the cytomegalovirus immediately early promoter IE-1 andintron A sequence and upstream of the bovine growth hormone poly-Asequence. The plasmid expresses SEAP in a constitutive manner whentransfected into mammalian cells.

Cloning of a Myristylated viral-H-ras expression plasmid

A DNA fragment containing viral-H-ras can be PCRed from plasmid “H-1”(Ellis R. et al. J. Virol. 36, 408, 1980) or “HB-11 (deposited in theATCC under Budapest Treaty on August 27, 1997, and designated ATCC209,218) using the following oligos.

Sense strand:

5′TCTCCTCGAGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAGCGCCGGATGACAGAATACAAGCTTGTGGTGG 3′. (SEQ.ID.NO.: 9)

Antisense:

5′CACATCTAGATCAGGACAGCACAGACTTGCAGC 3′. (SEQ.ID.NO.: 10)

A sequence encoding the first 15 aminoacids of the v-src gene,containing a myristylation site, is incorporated into the sense strandoligo. The sense strand oligo also optimizes the ‘Kozak’ translationinitiation sequence immediately 5′ to the ATG start site. To preventprenylation at the viral-ras C-terminus, cysteine 186 would be mutatedto a serine by substituting a G residue for a C residue in theC-terminal antisense oligo. The PCR primer oligos introduce an XhoI siteat the 5′ end and a XbaI site at the 3'end. The XhoI-XbaI fragment canbe ligated into the mammalian expression plasmid pCI (Promega) cut withXhoI and XbaI. This results in a plasmid in which the recombinantmyr-viral-H-ras gene is constitutively transcribed from the CMV promoterof the pCI vector.

Cloning of a viral-H-ras-CVLL expression plasmid

A viral-H-ras clone with a C-terminal sequence encoding the amino acidsCVLL can be cloned from the plasmid “H-1” (Ellis R. et al. J. Virol. 36,408, 1980) or “HB-11 (deposited in the ATCC under Budapest Treaty onAug. 27, 1997, and designated ATCC 209,218) by PCR using the followingoligos.

Sense strand:

5′TCTCCTCGAGGCCACCATGACAGAATACAAGCTTGTGGTGG-3′ (SEQ.ID.NO.: 11)

Antisense strand:

5′ CACTCTAGACTGGTGTCAGAGCAGCACACACTTGCAGC-3′ (SEQ.ID.NO.: 12)

The sense strand oligo optimizes the ‘Kozak’ sequence and adds an XhoIsite. The antisense strand mutates serine 189 to leucine and adds anXbaI site. The PCR fragment can be trimmed with XhoI and XbaI andligated into the XhoI-XbaI cut vector pCI (Promega). This results in aplasmid in which the mutated viral-H-ras—CVLL gene is constitutivelytranscribed from the CMV promoter of the pCI vector.

Cloning of c-H-ras-Leu61 expression plasmid

The human c-H-ras gene can be PCRed from a human cerebral cortex cDNAlibrary (Clontech) using the following oligonucleotide primers.

Sense strand:

5′-GAGAGAATTCGCCACCATGACGGAATATAAGCTGGTGG-3′ (SEQ.ID.NO.: 13)

Antisense strand:

5′-GAGAGTCGACGCGTCAGGAGAGCACACACTTGC-3′ (SEQ.ID.NO.: 14)

The primers will amplify a c-H-ras encoding DNA fragment with theprimers contributing an optimized ‘Kozak’ translation start sequence, anEcoRI site at the N-terminus and a Sal I stite at the C-terminal end.After trimming the ends of the PCR product with EcoRI and Sal I, thec-H-ras fragment can be ligated ligated into an EcoRI —Sal I cutmutagenesis vector pAlter-1 (Promega). Mutation of glutamine-61 to aleucine can be accomplished using the manufacturer's protocols and thefollowing oligonucleotide:

5′-CCGCCGGCCTGGAGGAGTACAG-3′ (SEQ.ID.NO.: 15)

After selection and sequencing for the correct nucleotide substitution,the mutated c-H-ras-Leu61 can be excised from the pAlter-1 vector, usingEcoRI and Sal I, and be directly ligated into the vector pCI (Promega)which has been digested with EcoRI and Sal I. The new recombinantplasmid will constitutively transcribe c-H-ras-Leu61 from the CMVpromoter of the pCI vector.

Cloning of a c-N-ras-Val-12 expression plasmid

The human c-N-ras gene can be PCRed from a human cerebral cortex cDNAlibrary (Clontech) using the following oligonucleotide primers.

Sense strand:

5′-GAGAGAATTCGCCACCATGACTGAGTACAAACTGGTGG-3′ (SEQ.ID.NO.: 16)

Antisense strand:

5′-GAGAGTCGACTTGTTACATCACCACACATGGC-3′ (SEQ.ID.NO.: 17)

The primers will amplify a c-N-ras encoding DNA fragment with theprimers contributing an optimized ‘Kozak’ translation start sequence, anEcoRI site at the N-terminus and a Sal I stite at the C-terminal end.After trimming the ends of the PCR product with EcoRI and Sal I, thec-N-ras fragment can be ligated into an EcoRI -Sal I cut mutagenesisvector pAlter-1 (Promega). Mutation of glycine-12 to a valine can beaccomplished using the manufacturer's protocols and the followingoligonucleotide:

5′-GTTGGAGCAGTTGGTGTTGGG-3′ (SEQ.ID.NO.: 18)

After selection and sequencing for the correct nucleotide substitution,the mutated c-N-ras-Val-12 can be excised from the pAlter-1 vector,using EcoRI and Sal I, and be directly ligated into the vector pCI(Promega) which has been digested with EcoRI and Sal I. The newrecombinant plasmid will constitutively transcribe c—N-ras-Val-12 fromthe CMV promoter of the pCI vector.

Cloning of a c-K-ras-Val-12 expression plasmid

The human c-K-ras gene can be PCRed from a human cerebral cortex cDNAlibrary (Clontech) using the following oligonucleotide primers.

Sense strand:

5′-GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3′ (SEQ.ID.NO.: 19)

Antisense strand:

5′-CTCTGTCGACGTATTTACATAATTACACACTTTGTC-3′ (SEQ.ID.NO.: 20)

The primers will amplify a c-K-ras encoding DNA fragment with theprimers contributing an optimized ‘Kozak’ translation start sequence, aKpnI site at the N-terminus and a Sal I stite at the terminal end. Aftertrimming the ends of the PCR product with Kpn I and Sal I, the c-K-rasfragment can be ligated into a KpnI —Sal I cut mutagenesis vectorpAlter-1 (Promega). Mutation of cysteine-12 to a valine can beaccomplished using the manufacturer's protocols and the followingoligonucleotide:

5′-GTAGTTGGAGCTGTTGGCGTAGGC-3′ (SEQ.ID.NO.: 21)

After selection and sequencing for the correct nucleotide substitution,the mutated c-K-ras-Val-12 can be excised from the pAlter-1 vector,using KpnI and Sal I, and be directly ligated into the vector pCI(Promega) which has been digested with KpnI and Sal I. The newrecombinant plasmid will constitutively transcribe c-K-ras-Val-12 fromthe CMV promoter of the pCI vector.

SEAP assay

Human C33A cells (human epitheial carcenoma—ATTC collection) are seededin 10 cm tissue culture plates in DMEM +10% fetal calf serum+1×Pen/Strep+1× glutamine+1× NEAA. Cells are grown at 37° C. in a 5% CO₂atmosphere until they reach 50 -80% of confluency.

The transient transfection is performed by the CaPO₄ method (Sambrook etal., 1989). Thus, expression plasmids for H-ras, N-ras, K-ras, Myr-rasor H-ras-CVLL are co-precipitated with the DSE-SEAP reporter construct.For 10 cm plates 600μl of CaCl₂ -DNA solution is added dropwise whilevortexing to 600 μl of 2× HBS buffer to give 1.2 ml of precipitatesolution (see recipes below). This is allowed to sit at room temperaturefor 20 to 30 minutes. While the precipitate is forming, the media on theC33A cells is replaced with DMEM (minus phenol red; Gibco cat.#31053-028)+0.5% charcoal stripped calf serum +1× (Pen/Strep, Glutamineand nonessential aminoacids). The CaPO4-DNA precipitate is addeddropwise to the cells and the plate rocked gently to distribute. DNAuptake is allowed to proceed for 5-6 hrs at 37° C. under a 5% C02atmosphere.

Following the DNA incubation period, the cells are washed with PBS andtrypsinized with 1 ml of 0.05% trypsin. The 1 ml of trypsinized cells isdiluted into 10 ml of phenol red free DMEM +0.2% charcoal stripped calfserum +1× (Pen/Strep, Glutamine and NEAA). Transfected cells are platedin a 96 well microtiter plate (10041/well) to which drug, diluted inmedia, has already been added in a volume of 100 μl. The final volumeper well is 200μl with each drug concentration repeated in triplicateover a range of half-log steps.

Incubation of cells and drugs is for 36 hrs at 37° C. under CO₂. At theend of the incubation period, cells are examined microscopically forevidence of cell distress. Next, 100 μl of media containing the secretedalkaline phosphatase is removed from each well and transferred to amicrotube array for heat treatment at 65° C. for 1 hr to inactivateendogenous alkaline phosphatases (but not the heat stable secretedphosphatase).

The heat treated media is assayed for alkaline phosphatase by aluminescence assay using the luminescence reagent CSPD® (Tropix,Bedford, Mass.). A volume of 50 jl media is combined with 200 μl of CSPDcocktail and incubated for 60 minutes at room temperature. Luminesenceis monitored using an ML2200 microplate luminometer (Dynatech).Luminescence reflects the level of activation of the fos reporterconstruct stimulated by the transiently expressed protein.

DNA-CaPO₄ precipitate for 10 cm. plate of cells

Ras expression plasmid (1 μg/μ1) 10 μl DSE-SEAP Plasmid (1 μg/μl) 2 μlSheared Calf Thymus DNA (1 μg/μl) 8 μl 2M CaCl₂ 74 μl dH₂O 506 μl

2× HBS Buffer

280 mM NaCl

10 mM KCl

1.5 mM Na₂HPO₄ 2H₂O

12 mM dextrose

50 mM HEPES

Final pH=7.05

Luminesence Buffer (26 ml)

Assay Buffer 20 ml Emerald Reagent ™ (Tropix) 2.5 ml 100 mM homoarginine2.5 ml CSPD Reagent ® (Tropix) 1.0 ml

Assay Buffer

Add 0.05M Na₂CO₃ to 0.05M NaHCO₃ to obtain pH 9.5.

Make 1 mM in MgCl₂

Example 21

The processing assays employed are modifications of that described byDeClue et al [Cancer Research 51, 712-717, 1991].

K4B-Ras processing inhibition assay

PSN-1 (human pancreatic carcinoma) or viral-K4B-ras-transformed Rat1cells are used for analysis of protein processing. Subconfluent cells in100 mm dishes are fed with 3.5 ml of media (methionine-free RPMIsupplemented with 2% fetal bovine serum or cysteine-free/methionine-freeDMEM supplemented with 0.035 ml of 200 mM glutamine (Gibco), 2% fetalbovine serum, respectively)containing the desired concentration of testcompound, lovastatin or solvent alone. Cells treated with lovastatin(5-10 μM), a compound that blocks Ras processing in cells by inhibitinga rate-limiting step in the isoprenoid biosynthetic pathway, serve as apositive control. Test compounds are prepared as 1000× concentratedsolutions in DMSO to yield a final solvent concentration of 0.1%.Following incubation at 37° C. for two hours 204 μCi/ml [³⁵S]Pro-Mix(Amersham, cell labeling grade) is added.

After introducing the label amino acid mixture, the cells are incubatedat 37° C. for an additional period of time (typically 6 to 24 hours).The media is then removed and the cells are washed once with cold PBS.The cells are scraped into 1 ml of cold PBS, collected by centrifugation(10,000×g for 10 sec at room temperature), and lysed by vortexing in 1ml of lysis buffer (1% Nonidet P-40, 20 mM HEPES, pH 7.5, 150 mM NaCl, 1mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, 10 μg/ml AEBSF, 10 μg/mlaprotinin, 2 μg/ml leupeptin and 2 μg/ml antipain). The lysate is thencentrifuged at 15,000×g for 10 min at 4° C. and the supernatant saved.

For immunoprecipitation of Ki4B-Ras, samples of lysate supernatantcontaining equal amounts of protein are utilized. Protein concentrationis determined by the bradford method utilizing bovine serum albumin as astandard. The appropriate volume of lysate is brought to 1 ml with lysisbuffer lacking DTT and 8 μg of the pan Ras monoclonal antibody, Y13-259,added. The protein/antibody mixture is incubated on ice at 4° C. for 24hours. The immune complex is collected on pansorbin (Calbiochem) coatedwith rabbit antiserum to rat IgG (Cappel) by tumbling at 4° C. for 45minutes. The pellet is washed 3 times with 1 ml of lysis buffer lackingDTT and protease inhibitors and resuspended in 100 μl elution buffer (10mM Tris pH 7.4, 1% SDS). The Ras is eluted from the beads by heating at95° C. for 5 minutes, after which the beads are pelleted by briefcentrifugation (15,000×g for 30 sec. at room temperature).

The supernatant is added to 1 ml of Dilution Buffer 0.1% Triton X-100, 5mM EDTA, 50 mM NaCl, 10 mM Tris pH 7.4) with 2 fg Kirsten-ras specificmonoclonal antibody, c-Kras Ab-1 (Calbiochem). The secondprotein/antibody mixture is incubated on ice at 4° C. for 1-2 hours. Theimmune complex is collected on pansorbin (Calbiochem) coated with rabbitantiserum to rat IgG (Cappel) by tumbling at 4° C. for 45 minutes. Thepellet is washed 3 times with 1 ml of lysis buffer lacking DTT andprotease inhibitors and resuspended in Laemmli sample buffer. The Ras iseluted from the beads by heating at 95° C. for 5 minutes, after whichthe beads are pelleted by brief centrifugation. The supernatant issubjected to SDS-PAGE on a 12% acrylamide gel(bis-acrylamide:acrylamide, 1:100), and the Ras visualized byfluorography.

hDJ processing inhibition assay

PSN-1 cells are seeded in 24-well assay plates. For each compound to betested, the cells are treated with a minimum of seven concentrations inhalf-log steps. The final solvent (DMSO) concentration is 0.1%. Avehicle-only control is included on each assay plate. The cells aretreated for 24 hours at 37° C./5% CO₂.

The growth media is then aspirated and the samples are washed with PBS.The cells are lysed with SDS-PAGE sample buffer containing 5%2-mercaptoethanol and heated to 95° C. for 5 minutes. After cooling onice for 10 minutes, a mixture of nucleases is added to reduce viscosityof the samples.

The plates are incubated on ice for another 10 minutes. The samples areloaded onto pre-cast 8% acrylamide gels and electrophoresed at 15 mA/gelfor 3-4 hours. The samples are then transferred from the gels to PVDFmembranes by Western blotting.

The membranes are blocked for at least 1 hour in buffer containing 2%nonfat dry milk. The membranes are then treated with a monoclonalantibody to hDJ-2 (Neomarkers Cat. #MS-225), washed, and treated with analkaline phosphatase-conjugated secondary antibody. The membranes arethen treated with a fluorescent detection reagent and scanned on aphosphorimager.

For each sample, the percent of total signal corresponding to theunprenylated species of hDJ (the slower-migrating species) is calculatedby densitometry. Dose-response curves and EC₅₀ values are generatedusing 4-parameter curve fits in SigmaPlot software.

Example 22 Rap1 processing inhibition assay

Protocol A:

Cells are labeled, incubated and lysed as described in

Example 21

For immunoprecipitation of Rap1, samples of lysate supernatantcontaining equal amounts of protein are utilized. Protein concentrationis determined by the bradford method utilizing bovine serum albumin as astandard. The appropriate volume of lysate is brought to 1 ml with lysisbuffer lacking DTT and 2 μg of the Rapl antibody, Rapl/Krevl (121)(Santa Cruz Biotech), is added. The protein/antibody mixture isincubated on ice at 4° C. for 1 hour. The immune complex is collected onpansorbin (Calbiochem) by tumbling at 4° C. for 45 minutes. The pelletis washed 3 times with 1 ml of lysis buffer lacking DTT and proteaseinhibitors and resuspended in 100 μl elution buffer (10 mM Tris pH 7.4,1% SDS). The Rap 1 is eluted from the beads by heating at 95° C. for 5minutes, after which the beads are pelleted by brief centrifugation(15,000×g for 30 sec. at room temperature).

The supernatant is added to 1 ml of Dilution Buffer (0.1% Triton X-100,5 mM EDTA, 50 mM NaCl, 10 mM Tris pH 7.4) with 2 fg Rap1 antibody,Rapl/Krevl (121) (Santa Cruz Biotech). The second protein/antibodymixture is incubated on ice at 4° C. for 1-2 hours. The immune complexis collected on pansorbin (Calbiochem) by tumbling at 4° C. for 45minutes. The pellet is washed 3 times with 1 ml of lysis buffer lackingDTT and protease inhibitors and resuspended in Laemmli sample buffer.The Rap1 is eluted from the beads by heating at 95° C. for 5 minutes,after which the beads are pelleted by brief centrifugation. Thesupernatant is subjected to SDS-PAGE on a 12% acrylamide gel(bis-acrylamide:acrylamide, 1:100), and the Rap1 visualized byfluorography.

Protocol B:

PSN-1 cells are passaged every 3-4 days in 10 cm plates, splittingnear-confluent plates 1:20 and 1:40. The day before the assay is set up,5×10⁶ cells are plated on 15 cm plates to ensure the same stage ofconfluency in each assay. The media for these cells is RPM1 1640(Gibco), with 15% fetal bovine serum and 1×Pen/Strep antibiotic mix.

The day of the assay, cells are collected from the 15 cm plates bytrypsinization and diluted to 400,000 cells/ml in media. 0.5 ml of thesediluted cells are added to each well of 24-well plates, for a final cellnumber of 200,000 per well. The cells are then grown at 37° C.overnight.

The compounds to be assayed are diluted in DMSO in ½-log dilutions. Therange of final concentrations to be assayed is generally 0.1-100 μM.Four concentrations per compound is typical. The compounds are dilutedso that each concentration is 1000× of the final concentration (i.e.,for a 10 μM data point, a 10 mM stock of the compound is needed).

2 μL of each 1000× compound stock is diluted into 1 ml media to producea 2×stock of compound. A vehicle control solution (2/L DMSO to lmlmedia), is utilized. 0.5 ml of the 2×stocks of compound are added to thecells.

After 24 hours, the media is aspirated from the assay plates. Each wellis rinsed with 1 ml PBS, and the PBS is aspirated. 180 μL SDS-PAGEsample buffer (Novex) containing 5% 2-mercapto-ethanol is added to eachwell. The plates are heated to 100° C. for 5 minutes using a heat blockcontaining an adapter for assay plates. The plates are placed on ice.After 10 minutes, 20 μL of an RNAse/DNase mix is added per well. Thismix is 1 mg/ml DNaseI (Worthington Enzymes), 0.25 mg/ml Rnase A(Worthington Enzymes), 0.5M Tris-HCl pH8.0 and 50 mM MgCl₂. The plate isleft on ice for 10 minutes. Samples are then either loaded on the gel,or stored at −70° C. until use.

Each assay plate (usually 3 compounds, each in 4-point titrations, pluscontrols) requires one 15-well 14% Novex gel. 25 μl of each sample isloaded onto the gel. The gel is run at 15 mA for about 3.5 hours. It isimportant to run the gel far enough so that there will be adequateseparation between 21 kd (Rap1) and 29 kd (Rab6).

The gels are then transferred to Novex pre-cut PVDF membranes for 1.5hours at 30 V (constant voltage). Immediately after transferring, themembranes are blocked overnight in 20 ml Western blocking buffer (2%nonfat dry milk in Western wash buffer (PBS +0.1% Tween-20). If blockedover the weekend, 0.02% sodium azide is added. The membranes are blockedat 4° C. with slow rocking.

The blocking solution is discarded and 20 ml fresh blocking solutioncontaining the anti Rap1a antibody (Santa Cruz Biochemical SC1482) at1:1000 (diluted in Western blocking buffer) and the anti Rab6 antibody(Santa Cruz Biochemical SC310) at 1:5000 (diluted in Western blockingbuffer) are added. The membranes are incubated at room temperature for 1hour with mild rocking. The blocking solution is then discarded and themembrane is washed 3 times with Western wash buffer for 15 minutes perwash. 20 ml blocking solution containing 1:1000 (diluted in Westernblocking buffer) each of two alkaline phosphatase conjugated antibodies(Alkaline phosphatase conjugated Anti-goat IgG and Alkaline phosphataseconjugated anti-rabbit IgG [Santa Cruz Biochemical]) is then added. Themembrane is incubated for one hour and washed 3× as above.

About 2 ml per gel of the Amersham ECF detection reagent is placed on anoverhead transparency (ECF) and the PVDF membranes are placed face-downonto the detection reagent. This is incubated for one minute, then themembrane is placed onto a fresh transparency sheet.

The developed transparency sheet is scanned on a phosphorimager and theRapla Minimum Inhibitory Concentration is determined from the lowestconcentration of compound that produces a detectable Rap1a Westernsignal. The Rap1a antibody used recognizes only unprenylated/unprocessedRap1a, so that the precence of a detectable Rap1a Western signal isindicative of inhibition of Rap1a prenylation.

Protocol C

This protocol allows the determination of an EC₅₀ for inhibition ofprocessing of Rap1a. The assay is run as described in Protocol B withthe following modifications. 20 μl of sample is run on pre-cast 10-20%gradient acrylamide mini gels (Novex Inc.) at 15 mA/gel for 2.5-3 hours.Prenylated and unprenylated forms of Rap1a are detected by blotting witha polyclonal antibody (Rap1/Krev-1 Ab#121; Santa Cruz Research Products#sc-65), followed by an alkaline phosphatase-conjugated anti-rabbit IgGantibody. The percentage of unprenylated Rap1a relative to the totalamount of Rap1a is determined by peak integration using Imagequant6software (Molecular Dynamics). Unprenylated Rap1a is distinguished fromprenylated protein by virtue of the greater apparent molecular weight ofthe prenylated protein. Dose-response curves and EC₅₀ values aregenerated using 4-parameter curve fits in SigmaPlot software.

Example 23 In Vivo Tumor Growth Inhibition Assay (nude mouse)

In vivo efficacy as an inhibitor of the growth of cancer cells may beconfirmed by several protocols well known in the art. Examples of suchin vivo efficacy studies are described by N. E. Kohl et al. (NatureMedicine, 1:792-797 (1995)) and N. E. Kohl et al. (Proc. Nat. Acad. Sci.U.S.A., 91:9141-9145 (1994)).

Rodent fibroblasts transformed with oncogenically mutated human Ha-rasor Ki-ras (10⁶ cells/animal in 1 ml of DMEM salts) are injectedsubcutaneously into the left flank of 8-12 week old female nude mice(Harlan) on day 0. The mice in each oncogene group are randomly assignedto a vehicle, compound or combination treatment group. Animals are dosedsubcutaneously starting on day 1 and daily for the duration of theexperiment. Alternatively, the farnesyl-protein transferase inhibitormay be administered by a continuous infusion pump. Compound, compoundcombination or vehicle is delivered in a total volume of 0.1 ml. Tumorsare excised and weighed when all of the vehicle-treated animalsexhibited lesions of 0.5-1.0 cm in diameter, typically 11-15 days afterthe cells were injected. The average weight of the tumors in eachtreatment group for each cell line is calculated.

21 1 4 PRT Artificial Sequence completely synthetic sequence 1 Cys ValLeu Ser 1 2 15 PRT Artificial Sequence completely synthetic sequence 2Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 1 5 10 15 352 DNA Artificial Sequence completely synthetic sequence 3 gagagggaattcgggccctt cctgcatgct gctgctgctg ctgctgctgg gc 52 4 41 DNA ArtificialSequence completely synthetic sequence 4 gagagagctc gaggttaacccgggtgcgcg gcgtcggtgg t 41 5 42 DNA Artificial Sequence completelysynthetic sequence 5 gagagagtct agagttaacc cgtggtcccc gcgttgcttc ct 42 643 DNA Artificial Sequence completely synthetic sequence 6 gaagaggaagcttggtaccg ccactgggct gtaggtggtg gct 43 7 27 DNA Artificial Sequencecompletely synthetic sequence 7 ggcagagctc gtttagtgaa ccgtcag 27 8 27DNA Artificial Sequence completely synthetic sequence 8 gagagatctcaaggacggtg actgcag 27 9 86 DNA Artificial Sequence completely syntheticsequence 9 tctcctcgag gccaccatgg ggagtagcaa gagcaagcct aaggaccccagccagcgccg 60 gatgacagaa tacaagcttg tggtgg 86 10 33 DNA ArtificialSequence completely synthetic sequence 10 cacatctaga tcaggacagcacagacttgc agc 33 11 41 DNA Artificial Sequence completely syntheticsequence 11 tctcctcgag gccaccatga cagaatacaa gcttgtggtg g 41 12 38 DNAArtificial Sequence completely synthetic sequence 12 cactctagactggtgtcaga gcagcacaca cttgcagc 38 13 38 DNA Artificial Sequencecompletely synthetic sequence 13 gagagaattc gccaccatga cggaatataagctggtgg 38 14 33 DNA Artificial Sequence completely synthetic sequence14 gagagtcgac gcgtcaggag agcacacact tgc 33 15 22 DNA Artificial Sequencecompletely synthetic sequence 15 ccgccggcct ggaggagtac ag 22 16 38 DNAArtificial Sequence completely synthetic sequence 16 gagagaattcgccaccatga ctgagtacaa actggtgg 38 17 32 DNA Artificial Sequencecompletely synthetic sequence 17 gagagtcgac ttgttacatc accacacatg gc 3218 21 DNA Artificial Sequence completely synthetic sequence 18gttggagcag ttggtgttgg g 21 19 38 DNA Artificial Sequence completelysynthetic sequence 19 gagaggtacc gccaccatga ctgaatataa acttgtgg 38 20 36DNA Artificial Sequence completely synthetic sequence 20 ctctgtcgacgtatttacat aattacacac tttgtc 36 21 24 DNA Artificial Sequence completelysynthetic sequence 21 gtagttggag ctgttggcgt aggc 24

What is claimed is:
 1. A compound of the formula D:

wherein: R^(1a) and R^(1b) are independently selected from: a) hydrogen, b) —N(R¹⁰)₂, c) C₁-C₆ alkyl unsubstituted or substituted by phenyl, R¹⁰O—, or —N(R¹⁰)₂; R^(1c) is independently selected from: a) hydrogen, b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—, c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent on the substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ and R¹¹OC(O)—NR¹⁰—; R³ is selected from H and CH₃; R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substituted with one or more of: 1) aryl, 2) OR⁶, 3) SR^(6a), SO₂R^(6a), or 4)

 and R² and R³ are optionally attached to the same carbon atom; R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆ cycloalkyl, aryl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R^(6a) is selected from; C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted or substituted with; a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R⁸ is independently selected from: a) hydrogen, b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F, Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—, and c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—; R^(9a) is hydrogen or methyl; R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl and aryl; R¹¹ is independently selected from C₁-C₆ alkyl and aryl; A¹ is selected from: a bond, —CH═CH—, —C≡C—, —C(O)—, —C(O)NR¹⁰—, O, —N(R¹⁰)—, or S(O)m; A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—: A⁴ is selected from: bond and —O—; V is selected from: a) pyridinyl or quinolinyl, and b) aryl; Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring, wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substituted with one or two moieties selected from the following: a) C₁₋₄ alkoxy, b) NR⁶R⁷, c) C₃₋₆ cycloalkyl, d) —NR⁶C(O)R⁷, e) HO, f) —S(O)mR^(6a), g) halogen, h) perfluoroalkyl, and i) C₁₋₄ alkyl; C₇-C₁₀ multicyclic alkyl ring is selected from:

 aryl is selected from: phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl; m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; r is 0 to 5, and v is 0, 1, 2 or 3; or a pharmaceutically acceptable salt thereof.
 2. A compound of the formula E:

wherein: R^(1a) and R^(1b) are independently selected from: a) hydrogen, b) —N(R¹⁰ ₂, c) C₁-C₆ alkyl unsubstituted or substituted by phenyl R¹⁰O—, or —N(R¹⁰)₂; R^(1c) is independently selected from: a) hydrogen, b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—, c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent on the substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ and R¹¹OC(O)—NR¹⁰—; R³ is selected from H and CH₃; R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substituted with one or more of; 1) aryl, 2) OR⁶, 3) SR^(6a), SO₂R^(6a), or 4)

 and R² and R³ are optionally attached to the same carbon atom; R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆ cycloalkyl, and aryl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R^(6a) is selected from:C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R⁸ is independently selected from: a) hydrogen, b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F, Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—, and c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—, R^(9a) is hydrogen or methyl; R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl and aryl; R¹¹ is independently selected from C₁-C₆ alkyl and aryl; A¹ is selected from: a bond, —CH═CH—, —C≡C—, —C(O)—, —C(O)NR¹⁰O—, O, —N(R¹⁰)—, or S(O)m; A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—; A⁴ is selected from: bond and —O—; V is selected from: a) pyridinyl or quinolinyl, and b) aryl; Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring, wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substituted with one or two moieties selected from the following: a) C₁₋₄ alkoxy, b) NR⁶R⁷, c) C₃₋₆ cycloalkyl, d) —NR⁶C(O)R⁷, e) HO, f) —S(O)mR^(6a), g) halogen, h) perfluoroalkyl, and i) C₁₋₄ alkyl; C₇-CIO multicyclic alkyl ring is selected from:

 aryl is selected from: phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl; m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; r is 0 to 5 and v is 0, 1, 2 or 3; or a pharmaceutically acceptable salt thereof.
 3. The compound according to claim 1 of the formula F:

wherein: R^(1b) is independently selected from: a) hydrogen, b) —N(R¹⁰)₂, c) C₁-C₆ alkyl unsubstituted or substituted by phenyl R¹⁰O—, or —N(R¹⁰)₂; R^(1c) is independently selected from: a) hydrogen, b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ or R¹¹OC(O)NR¹⁰—, c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent on the substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ and R¹¹OC(O)—NR¹⁰—; R³ is selected from H and CH₃; R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substituted with one or more of: 1) aryl, 2) OR⁶, 3) SR^(6a), SO₂R^(6a), or 4)

 and R² and R³ are optionally attach to the same carbon atom; R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆ cycloalkyl, and aryl, unsubstituted or substituted with a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R⁸ is independently selected from: a) hydrogen, b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F, Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—, and c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—; R^(9a) is hydrogen or methyl; R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl and aryl; R¹¹ is independently selected from C₁-C₆ alkyl and aryl; A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—; A⁴ is selected from: bond and —O—; Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring, wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substituted with one or two moieties selected from the following: a) C₁₋₄ alkoxy, b) NR⁶R⁷, c) C₃₋₆ cycloalkyl, d) —NR⁶C(O)R⁷, e) HO, f) —S(O)mR^(6a), g) halogen, h) perfluoroalkyl, and i) C₁₋₄ alkyl; C₇-C₁₀ multicyclic alkyl ring is selected from:

 aryl is selected from: phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl; m is 0, 1 or 2; p is 1, 2 or 3; r is 0 to 5, and v is 0, 1, 2 or 3; or a pharmaceutically acceptable salt thereof.
 4. The compound according to claim 2 of the formula G:

wherein: R^(1b) is independently selected from: a) hydrogen, b) —N(R¹⁰)₂, c) C₁-C₆ alkyl unsubstituted or substituted by phenyl R¹⁰O—, or —N(R¹⁰)₂; R^(1c) is independently selected from: a) hydrogen, b) R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, NO₂, R¹⁰C(O)—, N₃, —N(R¹⁰)₂or R¹¹OC(O)NR¹⁰—, c) unsubstituted or substituted C₁-C₆ alkyl wherein the substitutent on the substituted C₁-C₆ alkyl is selected from R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂NC(O)—, R¹⁰ ₂N—C(NR¹⁰)—, CN, R¹⁰C(O)—, N₃, —N(R¹⁰)₂ and R¹⁰OC(O)—NR¹⁰—; R³ is selected from H and CH₃; R² is selected from H;

 or C₁₋₅ alkyl, unbranched or branched, unsubstituted or substituted with one or more of: 1) aryl, 2) OR⁶, 3) SR^(6a), SO₂R^(6a), or 4)

 and R² and R³ are optionally attached to the same carbon atom; R⁶ and R⁷ are independently selected from: H; C₁₋₄ alkyl, C₃₋₆ cycloalkyl, and aryl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or c) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R^(6a) is selected from: C₁₋₄ alkyl or C₃₋₆ cycloalkyl, unsubstituted or substituted with: a) C₁₋₄ alkoxy, b) halogen, or ) phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl or isothiazolyl; R⁸ is independently selected from: a) hydrogen, b) C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ perfluoroalkyl, F, Cl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, CN, NO₂, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—, and c) C₁-C₆ alkyl substituted by C₁-C₆ perfluoroalkyl, R¹⁰O—, R¹⁰C(O)NR¹⁰—, (R¹⁰)₂N—C(NR¹⁰)—, R¹⁰C(O)—, —N(R¹⁰)₂, or R¹¹OC(O)NR¹⁰—; R^(9a) is hydrogen or methyl; R¹⁰ is independently selected from hydrogen, C₁-C₆ alkyl, benzyl and aryl; R¹¹ is independently selected from C₁-C₆ alkyl and aryl; A³ is selected from: —C(O)—, —C(O)NR¹⁰— or —C(O)O—; A⁴ is selected from: bond and —O—; Z is an unsubstituted or substituted C₇-C₁₀ multicyclic alkyl ring, wherein the substituted C₇-C₁₀ multicyclic alkyl ring is substituted with one or two moieties selected from the following: a) C₁₋₄ alkoxy, b) NR⁶R⁷, c) C₃₋₆ cycloalkyl, d) —NR⁶C(O)R⁷, e) HO, f) S(O)mR^(6a), g) halogen, h) perfluoroalkyl, and i) C₁₋₄ alkyl; C₇-C₁₀ multicyclic alkyl ring is selected from:

 aryl is selected from: phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl; m is 0, 1 or 2; p is 1, 2 or 3; r is 0 to 5, and v is 0, 1, 2 or 3; or a pharmaceutically acceptable salt thereof.
 5. A compound which is selected from: 1-(4-Cyanobenzyl)-5-[1-(2-oxo-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole R/S 1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole 1-(4-Cyanobenzyl)-5-[1-(2-hydroxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole 1-(4-Cyanobenzyl)-5-[1-(2-acetyloxy-2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]piperazine-4-(N-1-adamantyl)carboxamide 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(N-1-adamantyl)carbonyl piperazine 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-[N-(1R)-(−)-10-camphorsulfonyl]piperazine 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(1-adamantylmethyl) piperazine 1-(4-Cyanobenzyl)-5-[1-(2-(adamant-1-yl)ethyl)-2-oxo-piperazin-4-yl-methyl]imidazole 1-(4-Cyanobenzyl)-5-[1-(1-(adamant-1-yl)methyl)-2-oxo-piperazin-4-yl-methyl]imidazole 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl] piperazine-4-carboxylic acid (2-norbornane)methyl ester 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-piperazine-4-carboxylic acid (2-norbornane)methyl ester 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(2-bicyclo-[2.2.2]-octylcarbonyl)piperazine 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-(2-norbornanecarbonyl)piperazine 1-[1-(4′-Cyanobenzyl)imidazol-5-ylmethyl]-4-cis/trans-(2,6,6-trimethylbicyclo [3.1.1]heptanecarbonyl)-piperazine 1-(4′-Cyanobenzyl)-2-methyl-imidazol-5-ylmethyl piperazine-4-(N-1-adamantyl)carboxamide or a pharmaceutically acceptable salt or optical isomer thereof.
 6. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of claim
 1. 7. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of claim
 5. 8. A pharmaceutical composition made by combining the compound of claim 1 and a pharmaceutically acceptable carrier.
 9. A process for making a pharmaceutical composition comprising combining a compound of claim 1 and a pharmaceutically acceptable carrier.
 10. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of claim
 2. 11. A method for treating cancer related to a mutation, which is selected from a mutation in the ras gene and a mutation in a protein that can regulate Ras activity, which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of claim
 6. 12. A method according to claim 11 wherein the cancer is characterized by a mutated K4B-Ras protein.
 13. A method for treating cancer related to a mutation, which is selected from a mutation in the ras gene and a mutation in a protein that can regulate Ras activity, which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of claim
 7. 14. A method according to claim 13 wherein the cancer is characterized by a mutated K4B-Ras protein.
 15. A method for treating cancer related to a mutation, which is selected from a mutation in the ras gene and a mutation in a protein that can regulate Ras activity, which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition of claim
 10. 16. A method according to claim 15 wherein the cancer is characterized by a mutated K4B-Ras protein.
 17. A pharmaceutical composition made by combining the compound of claim 2 and a pharmaceutically acceptable carrier.
 18. A process for making a pharmaceutical composition comprising combining a compound of claim 2 and a pharmaceutically acceptable canner. 